Carbohydrates

Why can carbohydrates have these properties: • the existence of at least

one and often two or more asymmetric centers • the ability to exist either
in linear or ring structures • the capacity to form polymeric structures via
glycosidic bonds • the potential to form multiple hydrogen bonds with
water or other molecules in their environment.

Simplest aldose: glyceraldehyde

Simplest ketose: dihydroxyacetone

Overview of Carbohydrates

 Definition: Carbohydrates are biomolecules composed of carbon

(C), hydrogen (H), and oxygen (O) with the general
formula Cx(H2O)yCx(H2O)y.

 Classification:

o Monosaccharides: Single sugar units (e.g., glucose,

fructose).

o Disaccharides: Two monosaccharides linked by a glycosidic

bond (e.g., sucrose, lactose).

o Polysaccharides: Long chains of monosaccharides (e.g.,

starch, cellulose).

2. Biochemical Roles of Carbohydrates

 Energy Source: Glucose is the primary energy source for cells.

 Energy Storage: Glycogen (in animals) and starch (in plants) store
energy.

 Structural Support: Cellulose (in plants) and chitin (in insects)

provide structural integrity.

 Protection and Recognition: Carbohydrates on cell surfaces play

roles in cell-cell recognition and immune responses.

3. Monosaccharides

 Definition: Simplest form of carbohydrates with 3-7 carbon atoms.

  Examples:

o Hexoses: Glucose, fructose, galactose (6 carbons).

o Pentoses: Ribose, deoxyribose (5 carbons).

 Functional Groups:

o Aldoses: Contain an aldehyde group (e.g., glucose).

o Ketoses: Contain a ketone group (e.g., fructose).

 Naming: Based on the number of carbons and functional groups

(e.g., aldohexose, ketopentose).

4. Disaccharides

 Formation: Two monosaccharides linked by a glycosidic bond via a

condensation reaction.

 Examples:

o Sucrose: Glucose + fructose.

o Lactose: Glucose + galactose.

o Maltose: Glucose + glucose.

5. Polysaccharides

 Types:

o Homopolysaccharides: Composed of one type of

monosaccharide (e.g., starch, glycogen, cellulose).

o Heteropolysaccharides: Composed of different

monosaccharides (e.g., proteoglycans).

 Functions:

o Starch: Energy storage in plants.

o Glycogen: Energy storage in animals.

o Cellulose: Structural component in plant cell walls.

o Chitin: Structural component in insect exoskeletons.

6. Key Concepts in Carbohydrate Structure

  Chirality: Monosaccharides with 4 different groups on a carbon
atom are chiral.

 Stereoisomers:

o Enantiomers: Non-superimposable mirror images (e.g., D-

glucose and L-glucose).

o Diastereomers: Stereoisomers that are not mirror images

(e.g., glucose and galactose).

o Epimers: Diastereomers that differ at only one chiral center

(e.g., glucose and mannose).

 D and L Isomers: Based on the configuration of the highest chiral

carbon (D = OH on the right, L = OH on the left).

 Highest chiral carbon: the carbon furthest away from the

carbonyl group

 Number of D and L stereoisomers: 2n, n = number of chiral

centres

 Horizontal substituents:

7. Cyclic Structures of Monosaccharides

 Cyclization: In aqueous solutions, monosaccharides form cyclic

structures (pyranose or furanose).

 Anomeric Carbon: The carbon derived from the carbonyl group (C1
in aldoses, C2 in ketoses) that becomes chiral upon cyclization.

 Anomers:

o α-anomer: OH group on the anomeric carbon is trans to the

CH2OH group.

o β-anomer: OH group on the anomeric carbon is cis to the

CH2OH group.

 Mutarotation: Interconversion between α and β anomers in

aqueous solution.

8. Representing Sugars

 Fischer Projection: 2D linear representation showing chiral center,

dentifies D/L isomers and stereochemistry, shows positions of OH
 groups at chiral xenre , chiral centres are vertical and horizontal
bonds . horizontal substituents come out the page, vertical
substituents go into the page.

 Haworth Projection: 2D cyclic representation showing ring

structure and stereochemistry, Shows stereochemical arrangement
of groups on ring (up/down)

 Chair Form: 3D representation of cyclic sugars, showing axial and

equatorial positions. It minimizes steric strain by positioning bulky
groups in the equatorial positions and smaller groups in axial
position

9. Drawing Cyclic Sugars

 Steps:

1. Number carbons in the Fischer projection.

2. Rotate the Fischer projection 90° clockwise.

3. Perform bond rotation on C5.

4. Close the ring by forming a hemiacetal bond between C1 and

C5.

 Pyranose vs. Furanose: Pyranose forms a 6-membered ring with

5C and 1O, while furanose forms a 5-membered ring with 4C and
1O.

 Pyranose formation: OH group on C5 (or another appropriate OH)

reacts with the carbonyl group (aldehyde or ketone), forming a
hemiacetal or hemiketal, creating the pyranose

 Furanose formation: When the C5-OH reacts with Ketone group,

OH group on C4 reacts with the carbonyl group

Which is the most electrophilic carbon in sugar: carbonyl carbon

Which -OH attacks the carbonyl carbon to form most stable ring:

• In glucose, C5-OH attacks C1 → pyranose (6)

• In fructose, C5-OH (pyranose) or C4-OH (furanose, 5)

Why C5-OH in glucose? • Ring stability → 6-membered rings have less
strain • Steric hindrance → C5-OH has an ideal orientation for ring closure

10. Key Takeaways

 Carbohydrates are essential for energy, structure, and cell

recognition.

 Monosaccharides are the building blocks of carbohydrates and can

exist in linear or cyclic forms.

 Chirality and stereochemistry are critical for understanding

carbohydrate structure and function.

 Cyclic forms of sugars (pyranose and furanose) are more stable in

aqueous solutions, with β-anomers being more stable due to less
steric hindrance.

Summaries

1. Monosaccharides

Monosaccharides are the simplest carbohydrates, with 3-7 carbon atoms.

They can be classified based on functional groups (aldoses or ketoses) and
the number of carbons (trioses, pentoses, hexoses). Monosaccharides are
chiral and can exist in D or L configurations. In aqueous solutions, they
cyclize to form pyranose or furanose rings, with α and β anomers.

2. Disaccharides and Polysaccharides

Disaccharides are formed by linking two monosaccharides via a glycosidic

bond. Polysaccharides are long chains of monosaccharides and serve as
energy storage (starch, glycogen) or structural components (cellulose,
chitin). Polysaccharides can be homopolysaccharides (one type of
monosaccharide) or heteropolysaccharides (different monosaccharides).

3. Stereochemistry and Cyclization

Stereochemistry is crucial for understanding carbohydrate structure.

Monosaccharides can form cyclic structures in aqueous solutions, with the
anomeric carbon being the center of stereoisomerism. The β-anomer is
more stable due to less steric hindrance, and mutarotation allows
interconversion between α and β forms.

Flashcards
1. Monosaccharides

 Q: What is a monosaccharide?

 A: A single sugar unit with 3-7 carbon atoms, e.g., glucose, fructose.

 Q: What is the difference between aldoses and ketoses?

 A: Aldoses have an aldehyde group, while ketoses have a ketone

group.

 Q: What is chirality in monosaccharides?

 A: A molecule is chiral if it has a non-superimposable mirror image,

typically due to a carbon bonded to 4 different groups.

2. Disaccharides

 Q: What is a disaccharide?

 A: Two monosaccharides linked by a glycosidic bond, e.g., sucrose,

lactose.

 Q: How is a glycosidic bond formed?

 A: Via a condensation reaction, where a water molecule is removed.

3. Polysaccharides

 Q: What is a polysaccharide?

 A: A long chain of monosaccharides, e.g., starch, cellulose.

 Q: What is the function of glycogen?

 A: Energy storage in animals.

Where would you find alpha glucose: in starch, glycogen.

Where would you find beta glucose: in cellulose

4. Stereochemistry

 Q: What are enantiomers?

  A: Non-superimposable mirror images, e.g., D-glucose and L-
glucose.

 Q: What is an epimer?

 A: Diastereomers that differ at only one chiral center, e.g., glucose

and mannose.

5. Cyclic Structures

 Q: What is the anomeric carbon?

 A: The carbon derived from the carbonyl group in the linear form,
which becomes chiral upon cyclization.

 Q: What is mutarotation?

 A: The interconversion between α and β anomers in aqueous

solution.

Drawing fischer to pyranose:

Step 1:Number Carbons in Fischer projection, C5 -OH will react with C=O
at C1

Step 2: Rotate the Fischer projection 90 degrees clockwise (i.e. on its

side), Groups on right point down Groups on left point up

Step 3: Perform Bond rotation on C5

• C5 -OH attacks C1 , breaking C=O bond forming C1 -O bond.

• C1 -O bond is protonated to give C1 -OH.

• 5 C points so pyranose ring.

• All bonds facing down go bottom of ring (OH of C2 , C4 ). All bonds

facing up go above the ring (OH of C3 and CH2OH of C5 ).

• Exception is C1 which can be α or β

Step 4: Closure of the ring

Drawing Fischer to Furanose:

Step 1: Number Carbons in Fischer Projection

 Identify the carbons in the Fischer projection.

 The C4 -OH will react with the C=O at C1 (instead of C5 -OH as in

pyranose formation).
Step 2: Rotate the Fischer Projection 90 Degrees Clockwise

 Rotate the Fischer projection 90 degrees clockwise (on its side).

 Groups on the right in the Fischer projection point down in the ring.

 Groups on the left in the Fischer projection point up in the ring.

Step 3: Perform Bond Rotation on C4

 C4 -OH attacks C1, breaking the C=O bond and forming a C1 -O

bond.

 The C1 -O bond is protonated to give C1 -OH.

 This forms a 5-membered ring (furanose).

Step 4: Assign Substituents to the Ring

 All bonds facing down in the Fischer projection go to the bottom of

the ring (e.g., OH of C2).

 All bonds facing up in the Fischer projection go to the top of the

ring (e.g., OH of C3 and CH2OH of C5).

 The C1 -OH can be either α (down) or β (up), depending on the

anomeric configuration.

Key Differences from Pyranose Formation:

1. The C4 -OH (not C5 -OH) reacts with the carbonyl carbon (C1).

2. The resulting ring is a 5-membered furanose (not a 6-membered

pyranose).

3. The C5 -CH2OH group remains outside the ring as a substituent.

Drawing Fischer to Furanose (using fructose):

Step 1: Number the carbons in the Fischer projection

 In the Fischer projection of fructose, identify the carbons. Fructose is

a ketose, so the carbonyl group (C=O) is at C2 (not C1 as in aldoses
like glucose).

 The C5 -OH will react with the C=O at C2 to form the furanose ring.

Step 2: Rotate the Fischer projection 90 degrees clockwise

 Rotate the Fischer projection 90 degrees clockwise so that it is on its

side.

 After rotation:
 o Groups on the right in the Fischer projection will point down.

o Groups on the left in the Fischer projection will point up.

Step 3: Perform bond rotation on C5

 The C5 -OH attacks the C=O at C2, breaking the double bond and
forming a new bond between C2 and O.

 The C2 -O bond is protonated to give a C2 -OH.

 This forms a 5-membered ring (furanose).

Step 4: Determine the orientation of substituents

 In the furanose ring:

o All groups that were facing down in the Fischer projection will
point below the ring.

o All groups that were facing up in the Fischer projection will

point above the ring.

 The C1 carbon (which is now part of the ring) can be either α or β:

o α: The -OH group at C2 is below the ring.

o β: The -OH group at C2 is above the ring.

Cyclisation process: carbonyl carbon are sp2 hybridised (trigonal planar

geometry), so when the hydroxyl attacks the carbonyl, it can do so from
either side, thus generating two different stereoisomers or ANOMERS

Two types of anomers: when the newly formed hydroxyl group is pushed
down = Alpha anomer/ when the newly formed hydroxyl group is pushed
up = Beta Anomer *FOR D SUGARS

α (Alpha): the configuration of a cyclic sugar where the oxygen on the

anomeric carbon is on the opposite face of the ring relative to the
substituent on the other carbon flanking the ring oxygen

β (beta): the configuration of a cyclic sugar where the oxygen on the

anomeric carbon is on the same face of the ring as the substituent on the
other carbon flanking the ring oxygen

When the hydroxyl group at the anomeric carbon is on the same side of a
Fischer projection as the oxygen atom at the highest numbered
asymmetric carbon, the configuration at the anomeric carbon is , as in -D-
glucose. When the anomeric hydroxyl is on the opposite side of the
Fischer projection, the configuration is, as in -D-glucopyranose

Anomer: two diastereomeric monosaccharides that are epimers at the

anomeric carbon (differ only in their configuration at one C, the anomeric
C). Described with the terms α (“alpha”) and β (“beta”).

The anomeric carbon: the carbon connected to the OR and OH,

In haeworth projection: if newly formed hydroxyl is an alpha anomer it is

trans to the CH2OH group, if the newly formed hydrozyl is an beta
anomer, it is cis to the CH2OH

Substituents drawn to the left in a Fischer projection are drawn above the
ring in the corresponding Haworth projection. Substituents drawn to the
right in a Fischer projection are below the ring in a Haworth projection.

Cyclisation exist in dynamic equilibrium

Why is beta anomer preferred over alpha: the hydroxyl group in beta
anomer is in equatorial position, less steric hindarance, more stable

Mutarotation: the shift to equilibrium values for two anomers. E.G glucose
is 2:1 or 36%:64% (At equilibrium, the linear aldehyde or ketone structure
is only a minor component of the mixture (generally much less than 1%).)

Why are d sugars persistent in nature: e because of the stereospecificity

of the enzymes that synthesize and metabolize these small molecules

What are hemiacetals: r. Alcohols react readily with aldehydes to form

What are Hemiketals: ketones can react with alcohols to form hemiketals

In general, the pyranose form is favoured over the furanose ring for
aldohexose sugars, although, as we shall see, furanose structures are
more stable for ketohexoses.
Of all the D-aldohexoses, D-glucose is the only one that can adopt a
conformation with all its bulky groups in an equatorial position. With this
advantage of stability, it may come as no surprise that D-glucose is the
most widely occurring organic group in nature and the central hexose in
carbohydrate metabolism.

Sugars with free anomeric carbon atoms are reasonably good reducing
agent, Such reactions convert the sugar to a sugar acids

As in proteins and nucleic acids, each individual unit in an oligosaccharide

is termed a residue

The end of the molecule containing the free anomeric carbon is called the
reducing end, and the other end is called the nonreducing end. In the case
of sucrose, both of the anomeric carbon atoms are substituted, that is,
neither has a free OH group. The substituted anomeric carbons cannot be
converted to the aldehyde configuration and thus cannot participate in the
oxidation–reduction reactions characteristic of reducing sugars. Thus,
sucrose is not a reducing sugar.

how do anomeric carbons reduce compounds: Anomeric carbons reduce

compounds because they can readily convert to an open-chain form with a
free aldehyde or ketone group, which acts as a reducing agent, allowing it
to donate electrons and reduce other molecules

Why is the benedicts test effective: The Benedict's test is effective

because it specifically detects the presence of reducing sugars by utilizing
a chemical reaction where the copper ions in the Benedict's reagent are
reduced to copper(I) oxide when exposed to a reducing sugar

Homodisaccharides: contains one monosaccharide type

This ability to form branched structures distinguishes polysaccharides

from proteins and nucleic acids, which occur only as linear polymers

each sugar residue carries several hydroxyls, one or more of which may
be an acceptor of glycosyl substituents

Polysaccharides function as storage materials, structural components, or

protective substances
Organisms store carbohydrates in the form of polysaccharides rather than
as monosaccharides to lower the osmotic pressure of the sugar reserves,
Because osmotic pressures depend only on numbers of molecules, the
osmotic pressure is greatly reduced by formation of a few polysaccharides

Function of polysaccharides: energy storage, structure and protection,

recognition

Energy polysaccharides: starch, glycogen

Structure and protection polysaccharides: chitin, cellulose and

peptidoglycan

Recognition: Glycoproteins

Startch in plants: 70% amylopectin and 30% amylose

Amylopectin structure and function: compose of a-1-4-linked glucan

chains with small amounts of a-1-6-branch points , branched every 12-3
residues , many non-reducing ends but one reducing end makes up the
structural framework in starch

Amylose structure: linear and unbranched, contains one non-reducing

end and one reducing end  fills spaces within amylopectin matrix,
contributing to starch granule density

Why does amylopectin having many non-reducing ends beneficial: Starch

phosphorylase catalyses phosphorolytic cleavage of terminal glucose units
from the non-reducing end of the chain

Glycogen structure: glucose units linked b a(1,) glycosidic bonds with

a(1,6) branches every 8-12 residues

Cellulose function: structural support in plants

Cellulose structure: Cellulose is a linear homopolymer of D-glucose units

Difference between amylose and cellulose: cellulose contains b(1,4)

linkages

Chitin function: structural support in fungi, arthropods

Chitin structure: N-acetyl-b-D-glucosamine units lined by b(1,4) glycosidic

bonds, linear, forms fibres with H-bonding
Peptidoglycan function: polysaccharide of bacterial cell walls

Peptidoglycan structure: consists of N-acetyl-muramic acid (NAM) and N-

acetyl-glucosamine (NAG) in b(1,4) linkage

NAG composition: b-d-glucose + N-acetyl group (-NHCOCH3)

NAM composition: NAG + lactyl group (-CH(OH)-CH3) and a pentapeptide

chain

What is the function of NAM’s pentapeptide chain: essential for

peptidoglycan cross-linking

What forms the bacterial cell wall backbone: NAG-NAM linked b(1,4)bonds

Difference between gram – and gram + :

Glycoprotein structure: proteins with short, branched carbohydrate chains

attached (N- or O- linked)

Function of glycoproteins: cell recognition, signalling, and immune

response

Proteoglycan structure: proteins with long linear glycosaminoglycan

chains

Functions of proteoglycans: structural support in the extracellular matric,

hydration, and growth factor regulation

Differences between glycoproteins and proteoglycans:

Structure of GAGS : linear polysaccharides with repeating disaccharide
units, these disaccharides are acetylated amino sugars which have
negatively charged groups= acidic

Function of GAGS: components of glycoproteins and proteoglycans

How are glycoproteins formed: O-linked or N-linked glycosylation

What s glycosylation: the enzymatic addition of glycans (sugars) to

proteins

Where does glycosylation occur: ER and Golgi apparatus

O-linked Glycosylation

 Location: Occurs in the Golgi apparatus.

 Attachment: Glycans are attached to the oxygen atom (O) of the

serine (Ser) or threonine (Thr) side chain in proteins.

 Process:

o Sugars are added one at a time in the Golgi apparatus.

o No consensus sequence is required, but it often occurs in

regions rich in Ser, Thr, and proline.

N-linked: 1. N-linked Glycosylation

 Location: Occurs in the endoplasmic reticulum (ER) and Golgi

apparatus.

 Attachment: Glycans are attached to the nitrogen atom (N) of the

asparagine (Asn) side chain in proteins.

 Consensus Sequence: N-linked glycosylation occurs at the sequence

Asn-X-Ser/Thr, where X can be any amino acid except proline.
Function of glycoproteins: mucus production and protection, immune
response, blood group determination, cold adaptation, cell-cell signalling,
molecular transport

What is aggrecan: proteoglycan within cartilsiage

Wat is cartiliage: soft tissue between bones and joints

Why is aggrecan effective in cartilage: aggrecan is negatively charged,

associates strongly with H2O and acts as a lubricant

Function of proteoglycans: structural support in connective tissue,

carildae,a dn extracellular matrix

Structure of fatty acids: long non-polar tail, polar carboxyl head

How are fatty acids differentiated and named from another: length of
hydrocarbon chain, degree of unsaturation (number of double bonds)

Shorthand notation of FA:

Format: C:D (Δ position), C= Number of carbons, D= Number of double

bonds ,e.g. 18:1Δ(9) (Oleic acid: 18 carbons, 1 double bond at C-9)

Why does unsaturated fatty acid cause fluidity in lipids: double bonds
cause a “kink” (bend) in hydrocarbon tail, the kink produces flexible, fluids
aggregates and weaker bonds (easier for heat to access)

Why are saturated fatty acids less fluid: rigid arrays of saturated FA and
strong intermolecular bonds leave no room for motion

How does the conformation of unsaturated FA effects its fluidity: the

double bond in FA can either be in cis or trans. If in cis, the molecule has a
kink, if in trans it remains linear. Therefore double bonds in unsaturated FA
prefer to exist in cis conformation

How do humans produce longer fatty acid chain if palmitic acid is the
primary product of FA synthesis:

Elongation reactions: These reactions add two carbon unit sequentially to

the carboxyl ends of both saturated and unsaturated FA substrates
Where does this elongation reactions take place: In mitochondria and ER,
where source of carbon is acetyl-CoA or malonyl Co-A, respectively

Essential fatty acids vs conditionally essential fatty acids: Essential fatty

acids are fats that the human body cannot synthesize on its own and must
be obtained through the diet. Conditionally-essential fatty acids are fatty
acids that the body can synthesize under normal conditions

(EFA)Omega-3 Fatty Acids:

 Examples: Alpha-linolenic acid (ALA), Eicosapentaenoic acid

(EPA), and Docosahexaenoic acid (DHA).

(CEFA)Arachidonic Acid (AA):

 Normally synthesized from linoleic acid (an omega-6 fatty acid).

What can mammals not do in terms of FA production: •Mammals cannot

synthesize some polyunsaturated FAs •Mammals unable to introduce
double bonds in FA beyond C 9

How do mammals obtain linoleic acid: through their diets

Arachidonic acid synthesis through linoleic acid: The elongation adds 2C

atoms at carboxyl end (a decarboxylation step) which shifts the
C=C,*Process:

How are triglycerides: dehydration reaction,

glycerol + 3 fatty acids  ester (triglyceride + water)

function of triglycerides: adipocytes (pH stability), efficient energy source,

insulation

Why are triglycerides such a good energy source: meaning they store a
large amount of energy due to their structure containing long hydrocarbon
chains, this allows them to be efficiently stored in adipose tissue without
attracting excess water(anhydrous)

How do they produce metabolic water: Metabolic water can be produced

via FA oxidation (yields CO2 and H2O in TCA cycle)
Saponification: Reaction between TGs and NaOH or KOH yields free FA
salts and glycerol

FA salt behaviour in aqueous solution: In an aqueous solution, the FA salt

self-assembles and forms a micelle

Membrane

Function of plasma membrane:

(1) the exclusion of certain toxic ions and molecules from the cell,

(2) the accumulation of cell nutrients

(3) energy transduction

(4) cell locomotion,

(5) reproduction,

(6) signal transduction processes

(7) interactions with molecules or other cells in the vicinity.

Membrane impact on eukaryotes: endomembrane system allowed for

partitioning of labour within eukaryotic cells

What is the basic rationale for the formation of membranes: proteins and
lipids associate to form membranous structure (micelles) in water due to
hydrophobic effect and to increase entropy of system

What is the CMC: the concentration at which limits would predominantly

be monomers or micelles

How do composition of membranes suit their function: Membranes that

carry out many enzyme-catalyzed reactions and transport activities are
typically richer in protein, hereas membranes that carry out fewer protein-
related functions are richer in lipid.
Why do cell membranes adjust lipid and protein composition: Cellular
mechanisms adjust lipid and protein composition to functional needs

What are lipid bilayers: Lipid bilayers consist of back-to-back

arrangements of monolayer

Arrangement of lipid bilayers: The nonpolar portions of the lipids face the
middle of the bilayer, with the polar head groups arrayed on the bilayer
surface

Why do bilayers wrap around: Because exposure of the edges of the

bilayer to solvent is highly unfavourable, extensive bilayers normally wrap
around themselves and form closed vesicles

Why are these vesicles favourable: vesicles results in a favorable increase

in the entropy of the solution, because the water molecules are not
required to order themselves around the lipid chains

What does the hydrophobic core do to external ions and polar molecules:
This hydrophobic core provides a substantial barrier to ions and other
polar entities, thus they move slow across the membrane

What does the hydrophobic core do to non-polar/ hydrophobic molecules:

this same core also provides a favourable environment for nonpolar
molecules and hydrophobic proteins

What does the fluid mosaic model describe: Membrane bilayer is made up
of a mosaic of lipids, proteins (and carbohydrates) It is not a rigid or static
structure, instead it is fluid-like .Constituents that make up membrane are
capable of movement ,giving the dynamic ,fluid-like consistence

What are the three types: Phospholipids, glycolipids, cholesterol (sterols)

Types of phospholipids: glycerophospholipids, sphingolipids

Basic structure of a phospholipid:

 Hydrophobic tails: saturated fatty acid and unsaturated fatty acid

 Hydrophilic head: Glycerol (backbone molecule), phosphate
(attached to c3 in the glycerol)
Types of glycerophospholipids :

 Phosphate attached to the serine: phosphatidylserine

 Phosphate attached to the choline: phosphatidylcholine
 Phosphate attached to the ethanolamine: phosphatidylethanolamine

The phospholipase: enzymes that cleave bonds in glycerophospholipids

 PL-C and PL-D hydrolyse either side of the polar head (releasing
glycerol backbone and OH containing group respectively)
 PL-A1 and PL-A2 hydrolyse FA from C1 and C2 respectively

PL in snake venom: Phospholipase A2 in snake venom cause the release of

FA at C2 and ultimately breakdown of phospholipids

What is sphingosine: the backbone of sphingolipid

Composition of sphingosine: has an amino group and a unsaturated (18C)

hydrocarbon chain

Simplest sphingolipids: ceramides

What is the composition of ceramides: an amide linkage of 1 fatty acid to

the amino terminal of sphingosine

Function of ceramides: Signalling molecules, regulates structure,

differentiation & proliferation of cels

What is sphingomyelin: the dominant sphingolipid in nerve cell

membranes, major component of the myelin sheath

Glycolipids: glycolipids are ceramides bound to 1 or more sugar residues

in a glycosidic linkage

Types of glycolipids:

 Cerebrosides: contain a single sugar residue

 Gangliosides: contain multiple sugar residues

Structure of cholesterol : 4 fused hydrocarbon rings (3x 6-membered and

1x5 membered ring), one end of ring contains a hydrocarbon chain and
the other end has an OH group

How does cholesterol interact with the cell membrane: hydrophobic end is
embedded in membrane with FA chain, OH interacts with H2O and polar
heads oh phospholipids and sphingolipids
Why is the bilayer asymmetric and heterogenous: the two leaflets are not
structurally nor functionally identical

What is transverse asymmetry vs lateral heterogeneity:

Transverse asymmetry: refers to different lipid and protein composition

in the two leaflets of bilayer membrane

Lateral heterogeneity: refers to different lipid and protein compositions

in the plane of one of the leaflets of the membrane

3 processes that modulate movement of lipid in the membrane bilayer:

 Lateral diffusion
 Rotational diffusion
 Transverse diffusion (flip flop)

Difference in speeds of lipid movement: lateral and rotational movement

is very fast, transverse motion is very slow

Lipid-translocator protein function: “flip and flop” lipids across the

membrane bilayer

Type of lipid-translocator proteins:

 ATP-dependent flippases transport lipids from outer leaflets to inner

leaflet, Flippases = flip in (ATP required)
 ATP-dependent floppases transport lipids from inner leaflets to outer
leaflet,
Floppases = flop out (ATP required
 ATP-independent bi-directional scramblases moves lipids between

Scramblases = move both ways (no ATP, uses Ca² ⁺)

bilayers

What is the significance of the translocator proteins : These translocator

proteins help maintain membrane asymmetry and regulate lipid
composition

What are compartmentalised islands in cell membranes:

 Subdomains of the plasma membrane that contains high

concentrations of lipids and proteins
 Ordered and tightly packed, reduced fluidity
 Called lipid rafts
Function of these lipid rafts: serves to concentrate molecules to aid in
cellular processes such as cell signalling, signal transduction, protein
trafficking

Types of membrane proteins: peripheral and integral proteins

Peripheral proteins: includes those that do not penetrate the bilayer to

any significant degree and are associated with the membrane by virtue of
ionic interactions and hydrogen bonds between the membrane surface
and the surface of the protein

Integral proteins: possess hydrophobic surfaces that can readily penetrate

the lipid bilayer itself, as well as surfaces that prefer contact with the
aqueous medium. These proteins can either insert into the membrane or
extend all the way across the membrane and expose themselves to the
aqueous solvent on both sides

Types of integral proteins:

 integral polyprotic/transmembrane proteins span the entire width of

the membrane
 integral monoprotic proteins attached to one side of bilayer but do
not span entire width of membrane

characteristics of integral membrane proteins:

are permanently bound to membrane

difficult to isolate

can only be dissociated with strong detergent

How do peripheral proteins bind to membrane:

loosely

peripheral proteins form interactions and hydrogen bonds with polar

head groups of membrane lipids or with other integral proteins

they may interact with the non-polar membrane core by inserting a

hydrophobic loop or amphipathic a-helix

How do integral membrane proteins bind to the membrane:

firmly

the protein that are in contact with the non-polar core of the lipid
bilayer is dominated by a-helices and b-sheets
why are these proteins dominated by a-helices and b-sheets: these
secondary structures neutralize the highly polar NOH and CPO functions of
the peptide backbone through H-bond formation

What form does the anchored segment usually take shape in: a-helix

19-residue sequence in glycophorin: The glycophorin primary structure

consists of a segment of 19 hydrophobic amino acid residues with a short
hydrophilic sequence on one end and a longer hydrophilic sequence on
the other end. The 19-residue sequence is just the right length to span the
cell membrane if it is coiled in the shape of an -helix.

Beta sheets structure:

beta-sheets arrange in an antiparallel fashion forming b-barrel

Multiple B-sheets to make a barrel.

Need 9-11 amino acid per sheet to cross membrane

Hydrophobic aa orient toward the exterior and hydrophilic residues

orient toward the aqueous interior pore

Alpha helical structure:

a right-handed spiral formed by hydrogen bonds between the carbonyl

oxygen of one amino acid and the amide hydrogen of an amino acid
four residues away

Alpha-helical TM proteins may have a single TM segment or They may

have multiple TM segments 2-12

Hydrophobic Amino Acids Cross Membrane, need 21-25 aa to cross

membrane

Why does a-helix kink transmembrane proteins due to proline:

Membrane helix distortions are kinks at proline residues

Proline residues distorts the helix (it prevents H-bonds which straighten
the coil)

What do these kinks cause: proline induced kins create weak points in the
helix, which facilitate movement of plasma membrane
Hydropathy plots function: transmembrane a-helical domains can be
revealed by hydropathy plots based on its amino acid sequence

How does hydropathy plots work: The sequence is scanned 20 amino

acids at a time, and a mean hydrophobicity index is defined and plotted.
The hydropathy index is an average of the hydrophobicity values for the
20 residues

Example of integral membrane proteins (a-helix)

Glycophorin: a glycoprotein on red blood cells

Glycophorin structure: a single a-helical TM-segment protein

Glucose transporter: transport glucose across plasma membrane

Glucose transporter structure: A 12 TM-a helical segment

Example of integral membrane proteins (b-sheets)

Porins: Porins act as a "pore" through which certain molecules, such as

sugars, ions, and amino acids, can diffuse

Porins structure: 18 B-strands connected by loops, Polar residues face

inside and non-polar face the membrane

Advantages of B-strands over a-helices :

An α-helix requires 21-25 residues to cross the plasma membrane.

A β-strand requires only 9-11 residues to cross the plasma membrane.

Thus, with β-strands, a given amount of genetic material can encode a

larger number of transmembrane segments.

What 4 type of interactions are do peripheral proteins associate to the

membrane with:

Ionic and H-bond interactions

Amphipathic a-helix

Hydrophobic loop

Associated with integrated protein

Why are peripheral proteins easily dissociated from the membrane : due
to the weaker interaction, can dissociate from membrane

What is the function of lipid-anchoring in membrane proteins: proteins on

the surface of the bilayer are linked to lipid in membrane bilayer and this
anchors protein to membrane
What are lipid anchored proteins: is important in a variety of functions in
different cells and tissues. These proteins associate with membranes by
means of a variety of covalently linked lipid anchors.

Type of lipid-protein linkages:

N-Myristoylation

S-Palmitoylation

Prenylated anchors

Glycosylphosphatidylinositol (GPI) anchors

N-Myristoylation:

The fatty acid (FA) in N-myristoylation is always myristic acid (14:0).

Myristic acid forms an amide linkage with the glycine amino acid at the
N-terminal of the protein.

S-palmitoylation:

Palmitic acid (16:0) is linked via an ester linkage (COOH) to the –SH
group of a cysteine residue in the protein.

Serine and threonine residues can also be linked (though less

commonly than cysteine), and lipids such as palmitic acid, myristic
acid, and stearic acid can be used.

Prenylated anchors:

 Prenyl groups, such as farnesyl (15C:3) (farnesylation) and

geranylgeranyl (20C:4) (geranylgeranylation), are linked to the
carboxyl-terminal cysteine of proteins.

 The cysteine residue in the CaaX sequence of the protein

determines the type of prenylation.

o If X is alanine (Ala), methionine (Met), serine (Ser), or

glutamine (Gln), the protein will be farnesylated.

o If X is leucine (Leu), the protein will be geranylgeranylated.

 Aliphatic amino acids include alanine, glycine, valine, leucine,

isoleucine, phenylalanine, and proline.

Difference between myristoylation and prenylation/palmitoylation:

prenylation/palmitoylation are reversible, proteins can be attached and
detached from lipids
How does this detachment/attachment occur: Lipid modifications, such as
the attachment or release of lipids, provide a "switching device" for
altering the affinity of a protein to the membrane. This mechanism plays a
key role in modulating signal transduction pathways, as the addition or
removal of lipids can initiate or regulate cellular signalling processes.

Example of detachment in s-palmitoylation: enzyme Palmitoyl acyl

transferase binds cystine and fatty acid via an ester linkage, enzyme
palmitoyl thloesterase hydrolyses the ester linkage

Transport across membrane

How do cells maintain a very large amount of potential energy: in the form
of concentration gradients

What type of proteins are transport proteins: integral membrane proteins

What problem do transport proteins solve: the transfer of hydrophilic

substances across the hydrophobic membrane

Types of membrane transport processes: passive diffusion, facilitated

diffusion, and active transport

Process of movement in passive diffusion: , the transported species moves

across the membrane in the thermodynamically favoured direction
without the help of any specific transport system/molecule

Passive diffusion for charged vs uncharged molecule:

Uncharged molecule: passive diffusion is an entropic process, in which

movement of molecules across the membrane proceeds until the
concentration of the substance on both sides of the membrane is the
same

Charged species: For a charged species, the situation is slightly more

complicated. In this case, the movement of a molecule across a
membrane depends on its electrochemical potential.

Types of molecules in passive diffusion: small uncharged molecules (H2O,

O2)

Cons of passive diffusion: its too slow

Facilitated diffusion: proteins in the cell membranes that facilitate
transport of these species across the membrane

Types of molecules in facilitated diffusion: large or polar molecules (K+,

Na+, sugars)

Type of proteins used in facilitated diffusion: channel proteins and carrier

proteins

What do facilitate transport proteins have in common:

They facilitate net movement of solutes only in the thermodynamically

favoured direction

they display a measurable affinity and specificity for the transported

solute

what does facilitated and active transport have in common: they transport
species in selective manner

Facilitated diffusion of glucose:

glucose binds to GLUT1 transport , which is in its T1 conformation,

where its binding site is open to the outside of the cell

glucose binding of GLUT1 to change to its T2 conformation, where its

binding site is open to the inside of the cell

glucose is released to the interior of the cell, initiating a second

conformational change in GLUT1

Loss of bound glucose causes GLUT1 to return to its original (t1)

conformation, ready to repeat

What is active transport: a transport which requires energy input to move

substances against their concentration gradient

Types of energy input in active transport:

ATP hydrolysis

Light energy

Energy stored in ion gradients

What and why is the transport that depends on the ion gradient for its
energy input referred to as: secondary active transport/co -active
transport, The original ion gradient is said to arise from a primary active
transport process, and the transport that depends on the ion gradient for
its energy input
What is electrogenic transport: When transport results in a net movement
of electric charge across the membrane, if not it is electrically neutral

Energy coupling , with ATP, in active transport: depends on ATP hydrolysis

thus couples chemical free energy to mechanical (translational) free
energy

What is the function of Na+,K+-ATPase (sodium): accumulate K+ and

extrude Na+

How does sodium pump work: The sodium pump actively pumps three
Naions out of the cell and two K ions into the cell per ATP hydrolyzed

(ATP4-) + H2O + (3 Na+(inside))+ (2 K+(outside))⎯⎯→(ADP3 - )+ H2PO4 +

(3 Na+(outside) )+ (2 K+(inside))

Glucose co-active transport:

Glucose is bound and released inside cell against conc. gradient

Thus, the electrochemical energy released from Na+, because High

Na+ outside cell compared to inside, moving down gradient co-
transports glucose against its gradient

Symporters: with the ion and the transported amino acid or sugar moving
in the same direction as the ion going down its concentration gradient

Antiport: the ion and the other transported species move in opposite
directions

Nucleic Acids

Nucleotide composition: biological molecules that possess a heterocyclic

nitrogenous base, a five-carbon sugar (pentose), and phosphate as
principal components of their structure

What are nucleic acids: linear polymers of nucleotides

Sugars in dna and rna: 2-deoxyribose and ribose

What are pyrimidines: Pyrimidines are six-membered heterocyclic

aromatic rings containing two nitrogen atoms
What are purines: consists of two rings of atoms: one resembling the
pyrimidine ring and another resembling the imidazole ring (a five-
membered, heterocyclic, aromatic compound)

Purines: Adenine (6-amino purine) and Guanine (2-amino-6-oxy purine)

Pyrimidines: Cytosine (2-oxy-4-amino pyrimidine), Uracil (2-oxy-4-oxy

pyrimidine), Thymine (2-oxy-4-oxy 5-methyl pyrimidine)

What allows purine and pyrimidines to undergo keto-enol tautomeric

shifts: The aromaticity of the pyrimidine and purine ring systems and the
electron-rich nature of their carbonyl and ring nitrogen substituents endow
them with the capacity to undergo keto–enol tautomeric shifts

Relationship between pH and pKa:

If the pH is higher than the pKa, compound will be deprotonated

If the pH is lower than the pKa, compound will be protonated

What tautomer dominates in uracil, thymine and guanine at neutral pH:

the keto tautomer,

Why: pKa values for Nitrogen 1 and 3 are greater than 8

What tautomer dominates in cytosine at neutral pH: the enol form

dominates

Why: pKa value for N-3 in this pyrimidine is 4.5

What are the importance of the pk values of these bases: , they are
important in determining whether these nitrogenous bases serve as H-
bond donors or acceptors, which creates the hydrogen bonds between
bases.

What are the important functional groups that are involved in the
hydrogen bonding:. The important functional groups participating in H-
bond formation are the amino groups of cytosine, adenine, and guanine;
the ring nitrogens at position 3 of pyrimidines and position 1 of purines;
and the strongly electronegative oxygen atoms attached at position 4 of
uracil and thymine, position 2 of cytosine, and position 6 of guanine

Hyperchromic effect: the increase of absorbance of uv light when two DNA

strands separate
Why does this increase occur: due to the loss of stacking between dna
molecules

What are nucleosides: compounds formed when a base is linked to a sugar

What are the sugars of nucleosides: pentoses that exist as furanoses , d-

ribofuranose and 2-deoxy-d-ribofuranose

Benefits of furanose as the sugar in nucleosides:

Five-membered ring ensures stability, flexibility and efficient base

pairing

Furanose geometry supports DNA helix and RNA structural diversity

Characteristics of the bond between the sugar and the bases:

Linked via N-glycosidic bonds

Are always in B configuration

Why are nucleosides more hydrophilic than free bases: hydrophilicity of

the pentose

Conformation of nucleotide: syn and anti, the two possible orientation of a

base around the glycosidic bond

Favoured conformation of purines: can adopt both syn and anti, but anti
conformation is more favoured

Favoured conformation of pyrimidines: strongly favor anti-conformation,

because syn leads to steric clashes with the sugar

When does a nucleotide form: results when phosphoric acid is esterified to

a sugar, contain 5’ monophosphate groups

Why do nucleotides have acidic properties: . Because the pKa value for
the first dissociation of a proton from the phosphoric acid moiety is 1.0 or
less and the pKa for the second dissociation of a proton is 6, the net
charge on a nucleoside monophosphate is 2

What neutralises this negative charge in phosphate: Mg+2

Derivatives of nucleotides at the phosphate group :

Nucleoside diphosphate (NDP) and nucleoside triphosphate (NTP)

Cyclic nucleotides
What are the role of nucleotide 5’ triphosphate: they are carriers of
chemical energy

Function of NTPs:

ATP is central to energy metabolism

GTP drives protein synthesis

CTP drives lipid synthesis

UTP drives carbohydrate metabolism

Why are different NTP’s needed: bases act as recognition in cellular

processes

What are nucleic acids linked by: linear polymers of nucleotides linked 3’to
5’ by phosphodiester bridges

What gives the nucleic acid polymers there unique property: the nature of
the base

Where does the information to make all macromolecules of cell lie: in DNA

How is this information accessed: it is accessed through transcription

What did chargaff prove: that dna molecule is made up of base pairs,
where the base pairs are specific and always in equal ratio

What did Watson and crick prove: dna was a complementary double helix

Important of base pairing:

the separation between base pairs are equal, so double helix is

maintained equally

base pairing are complementary, Therefore, separation of the two

strands and faithful replication of each, through a process in which
base pairing specifies the nucleotide sequence in the newly
synthesized strand, leads to two progeny molecules identical in every
respect to the parental double helix

what is the difference between ribose and 2-deoxyribose:

Presence of 2’-OH makes RNA unstable (ie has a short half-life) thus
absence of 2’-OH makes DNA more stable

Benefit of RNA instability:

 A base (OH–) can attack and deprotonate the H+ from the 2’–OH and
so water is lost

This generates 2'–O– which can attack the phosphate of the

phosphodiester bond

The phosphorus then detaches from the oxygen that connects it to the
adjacent sugar, resulting in cleavage of the RNA backbone. Catalysed
by phosphodiesterase (nucleases)

This produces a 2’,3’-cyclic phosphodiester intermediate that can then

yield a 2’- or a 3’-phosphate when hydrolysed

How is uracil formed: deamination of cytosine to form a carbonyl bond

What does the conversion between C and U cause: could possibly result in
a heritable change of a C:G pair to a U:A pair

How do enzymes deal with mutant U: removes them and replaces them
with C

What if U was a natural U and not a mutation: enzymes replace this U with
5-methyl-U, known as thymine, so U in DNA is identified and removed

Composition of ribosomes: 2/3 RNA and 1/3 protein

rRNA function serves as a scaffold for ribosomal proteins

2 subunits in rRNA: large (lsu) and small (ssu)

Function of LSU: structural and catalytic functions in protein translation,

LSU has “APE” sites

Function of SSU: binds and holds mRNA

What causes rRNA secondary structure: High sequence complementarity

allows extensive intra-strand base-pairing leading to a highly folded
pattern

Why is folding pattern in ribosomes conserved across species:

Evolutionarily conserved rRNA secondary structure ensures all ribosomes
function similarly in protein synthesis

Secondary structure of tRNA:

High sequence complementarity allows

extensive intra-strand base-pairing –

creates 4 base-paired segments, three loops, and one stem.

tRNA linkage to amino acid residue: 3'-terminal sequence is always CCA-
linked to amino acid residue

function of anticodon loop: contains 3 unpaired bases which are the

anticodon (could be non-canonical bases)

Difference in transcription products of DNA in prokaryotes vs eukaryotes:

 In prokaryotes, a single mRNA contains the information for synthesis

of many proteins
 In eukaryotes, a single mRNA codes for just one protein, but needs
processing to remove introns, etc

What bonds stabilise DNA helix:

 Hydrogen bonds between complimentary bases and between sugar

phosphate backbone and H2O
 Electrostatic shield: Negatively charged phosphate groups repel one
another. Neutralised by interaction with Mg2+
 Stacking of base pair : Van der Waals and hydrophobic interactions

What is base pair stacking: To minimize contact with water, these bases
stack on top of each other, with their planes nearly parallel and surfaces
nearly in contact.

Characteristics of bonds between conical base pairs:

 A and T share two H-bonds.

 G and C share three H-bond
 Nearly identical distance between bonds
 G-C bonds are stronger

The two distinct grooves/cuts form in DNA: major and minor grooves that
run opposite each other

Major groove vs Minor groove:

 Major groove: top-edge of base-pair (‘base-pairing side”). Sugar

phosphate is far apart
 Minor groove: bottom-edge of base-pair (“no base-pairing side”).
Sugar phosphate is close together
 Major and minor are opp. each other
What do the grooves allow: Within each groove, base-pairs are accessible
to other molecules

Example of molecules accessing DNA grooves:

 DNA-binding-proteins use these to “read” a sequence to bind

 Regulatory proteins (transcription factors) can recognize the pattern
of bases and H bonding possibilities in the major groove

How can DNA be denatured:

 Heating DNA to >80oC or in alkali

 changes in pH (> pKa)

Tertiary structure of DNA: DNA secondary (2°) structure can form

supercoils for compaction

What enzymes are involved in the supercoiling: topoisomerases and

gyrases introduce or remove supercoil

Type of supercoiling: Negative and positive supercoiling

Negative supercoiling:

 Reduces the number of base pairs per turn

 Makes the DNA helix more accessible for processes like transcription
and replication by loosening the structure.

Positive supercoiling:

 Increases the number of base pairs per turn

 Makes the DNA helix tighter and more compact, which can hinder
processes like transcription and replication.

How is dna packaged in eukaryotic cells:

• Wrapped around histone proteins → forms nucleosomes

• Further packing of nucleosomes → forms chromosome

Nucleosomes structure:

• DNA wraps around histone octamers for compaction

• Histone core: 4 pairs of distinct histones (H2A, H2B, H3, H4), forming
a positively charged octamer
Chromosome structure: multiple nucleosomes (~6 per turn) resemble
beads on a string

What does DNA being semi-conservative mean: Each parental strand

remains associated with its newly synthesized complement, so each DNA
duplex contains one parental strand and one new strand

Why does DNA become semi-conservative: Each original strand of the

double helix serves as a template and is copied to form a new
complementary strand (DNA polymerase/s)

DNA replication initiation:

 Replication begins at specific regions called origins of replication

(Ori). In E. coli, this is known as oriC.

 Helicase unwinds the double-stranded DNA (dsDNA) by breaking

hydrogen bonds between base pairs, exposing single-stranded DNA
(ssDNA).

 Single-stranded binding proteins (SSBs) stabilize the ssDNA

and prevent the strands from re-annealing.

 Unwinding causes positive supercoiling ahead of the replication

fork. Gyrase (a type II topoisomerase) relieves this tension by
introducing negative supercoils using energy from ATP hydrolysis

DNA primer synthesis:

Primase synthesizes short RNA primers (~10 nucleotides) at the origin

and along the lagging strand. These primers provide a free 3’-OH group
for DNA Polymerase to begin synthesis.

DNA replication elongation:

 DNA Polymerase III (the main replicative enzyme in E. coli)

adds nucleotides in the 5’ to 3’ direction, using the parental
strands as templates.

 Replication is bidirectional, with two replication forks

moving in opposite directions away from the origin.

 Leading strand: Synthesized continuously in the 5’ to 3’

direction toward the replication fork.
  Lagging strand: Synthesized discontinuously away from the
replication fork as Okazaki fragments (short segments of
DNA). Each fragment begins with an RNA primer.

DNA replication termination:

 DNA Polymerase I removes the RNA primers and fills the gaps
with DNA.

 DNA ligase joins the Okazaki fragments on the lagging strand by

forming phosphodiester bonds, creating a continuous DNA strand.

DNA proofreading and repair:

 DNA Polymerase III and I have 3’ to 5’ exonuclease activity,

which allows them to proofread and correct errors during replication.

 Mismatch repair systems further ensure the accuracy of DNA

replication.

Enzymes involved :

 Helicase: Unwinds DNA.

 Gyrase: Relieves supercoiling.

 Primase: Synthesizes RNA primers.

 DNA Polymerase III: Main enzyme for DNA synthesis.

 DNA Polymerase I: Removes primers and fills gaps.

 DNA ligase: Joins Okazaki fragments.

Types of mutations in DNA:

Deamination, depurination, amin-imino and keto/enol automerism,

dimerization

Deamination mutation:

• Deamination of cytosine forms uracil - which base pairs with

adenine. C-G becomes U-A
• Deamination of adenine forms hypoxanthine (analogue of adenine) -
which base pairs with cytosine. A-T becomes C-G
Depurination mutation: The loss of purines (or pyrimidines) from DNA
resulting from hydrolysis of the glycosylic bond between deoxyribose and
the base, leaving an apurinic/apyramidic site (AP) site in DNA

Amini-imino tautomerism:

• Amino (-NH₂) → Imino (=NH) transformation due to deprotonation

• Adenine (amino) pairs with Thymine (A:T)
• Adenine (imino) mispairs with cytosine (A:C) → leads to mutations

Keto-enol tautomerism:

• Guanine (keto) normally pairs with cytosine (G:C)

• Guanine (enol) mispairs with Thymine (G:T) → causes base-pairing
error

Mutation in dimerization:

• UV light promotes the formation of covalent crosslinks between

adjacent pyrimidine residues in a DNA strand e.g. thymine residues
• A thymine dimer distorts the double helix, and so replication and
gene expression are impaired

DNA repair mechanisms:

Direct reversal, mismatch repair, base excision repair, nucleotide excision

Direct reversal:

Know as light repair, Photolyase detects and binds at the thymine dimer
and uses light

energy to break the dimer, restoring the pyrimidines to their original form

Mismatch repair :

Methyl-directed in [Link]

1. Damage Recognition:

o Prokaryotes: The MutS protein recognizes and binds to

mismatched base pairs or insertion-deletion loops.

o Eukaryotes: MSH proteins (e.g., MSH2-MSH6 or MSH2-

MSH3 complexes) perform the same function.

2. Strand Discrimination:
 o Prokaryotes: MutL interacts with MutS to identify the newly
synthesized strand (which contains the error). The parental
(template) strand is distinguished by transient nicks or
methylation patterns.

o Eukaryotes: MLH proteins (e.g., MLH1-PMS2 complex) work

with MSH proteins to target the nicked or newly synthesized
strand for repair.

3. Excision of Mismatched Segment:

o An exonuclease (e.g., EXO1 in eukaryotes) removes the

mismatched section of the DNA strand, creating a single-
stranded gap.

4. Resynthesis:

o DNA polymerase (e.g., DNA Polymerase δ in eukaryotes) fills

the gap by synthesizing new DNA, using the intact template
strand as a guide to ensure the correct bases are
incorporated.

5. Ligation:

o DNA ligase seals the nick in the repaired strand, restoring the
integrity of the DNA double helix.

Base excision repair (BER): Uracil containing DNA:

Complementary systems in [Link] and eukaryotes

1. Damage recognition: DNA glycosylase recognizes and removes the

damaged or incorrect base, creating an AP (apurinic/apyrimidinic) site

2. AP site cleavage: AP endonuclease cleaves the DNA backbone at the AP

site, generating a single- strand break

3. Gap processing: DNA polymerase (Pol β in eukaryotes) removes the

sugar-phosphate remnant and inserts the correct base

4. Ligation: DNA ligase (Ligase I or Ligase III) seals the nick, restoring DNA
integrity

Nucleotide excision repair (NER):

• Similar to BER but recognizes and repairs larger regions of damaged

DNA
• Nucleases cleave on the 3′ and 5′ sides of the damaged site, thus
excising an oligonucleotide

• The oligonucleotide spans 12 - 13 nucleotides in prokaryotes & 27 - 29

nucleotides in eukaryotes

• The gap is filled in by DNA polymerase

• Pol I in prokaryotes or Pol δ or ε in eukaryotes

• Sugar–phosphate backbone is covalently closed by DNA ligase

AMINO ACIDS

Basic structure of an amino acid: central alpha carbon connected to a

amino and carboxyl group, and a hydrogen and R side chain group.

Amino and carboxyl group at neutral pH: amino group is protonated and
carboxyl group is deprotonated. This is called a zwitterion

Amino acid stereochemistry: chiral molecules. With four different groups

attached to it, the -carbon is said to be asymmetric. The two possible
configurations for the -carbon constitute nonidentical mirror-image
isomers or enantiomers

What are peptide bonds: bonds that allow amino acids to polymerise,
which is the reaction between NH3+ and COO- in different amino acids to
form a covalent amid linkage, where water molecule is eliminated

What does the equilibrium for the reaction of amino acids favour: favours
peptide bond hydrolysis, thus peptide bond formation requires energy
input

How are the common amino acids classified:

 nonpolar or hydrophobic amino acids,

 neutral (uncharged) but polar amino acids,
 acidic amino acids (which have a net negative charge at pH 7.0),
and
 basic amino acids (which have a net positive charge at neutral pH)

Important roles that these amino acids play in biological systems:

  Non-polar amino acids: are critically important for the processes
that drive protein chains to “fold
 Polar amino acids: (1) form hydrogen bonds with water, and (2) play
a variety of nucleophilic roles in enzyme reactions
 Acidic amino acids: bind metal ions for structural or functional
purposes possess
 Basic amino acids: play important roles as proton donors

Why are amino acids considered weak polyprotic acids: The ionizable
groups are not strongly dissociating ones, and the degree of dissociation
thus depends on the pH of the medium. All the amino acids contain at
least two dissociable hydrogens.

How are the dissociation constants of both the a-carboxyl and a-amino
groups are affected by the presence of the other group:

The adjacent a-amino group makes the a-COOH group more acidic (that is,
it lowers the pKa), so it gives up a proton more readily than simple alkyl
carboxylic acids.

How does the amino do this: The a-NH3 + (ammonium) group is strongly
electron-withdrawing, and the positive charge of the amino group exerts a
strong field effect and stabilizes the carboxylate anion.

What is the isoelectric point: the pH at which the molecule has no net
charge

What is optical activity in enantiomeric molecules: the ability to rotate the

plane of polarization of plane polarized light

Dextrorotary (+): Clockwise rotation of incident light is referred to as

dextrorotatory behaviour

Levorotatory(-): d counterclockwise rotation is called levorotatory

behaviour

What does the magnitude and direction of the optical rotation depend on:
the nature of the amino acid side chain

why is measuring the absorbance of aromatic amino acids in peptide

chain significant:
these amino acids absorb UV light strongly, allowing for protein
quantification

why: The absorbance of aromatic amino acids at around 280 nm is directly

proportional to the protein concentration, making it a simple and widely
used method for estimating protein amounts in a sample, measuring UV
absorbance at 280nm (W) is a fast and convenient method for detection
and quantative analysis of proteins

Chromatography: the separation of mixtures dissolved in a mobile phase

(liquid,gas) on a stationary phase (liquid, solid).

What is the partition coefficient:

the ratio of the concentration of a substance in one medium or phase (C 1)

to the concentration in a second phase (C2) when the two concentrations
are at equilibrium (Any solute will partition between two immiscible
solvents)

what cause the separation in chromatography: occurs through differential

retention of compounds by the stationary phase based on parameters
such as surface absorption, solubility, size and charge

Ion exchange chromatography:

 Ion Exchange Chromatography Can Be Used to Separate Molecules

on the Basis of Charge
 Principle: which the charged molecules of interest (ions) are
exchanged for another ion (usually a salt ion) on a charged solid
support
 Example: solutes in a liquid phase, usually water, are passed
through a column filled with a porous solid phase composed of
synthetic resin particles containing charged groups. Resins
containing positively charged groups attract negatively charged
solutes and are referred to as anion exchange resins. Resins with
negatively charged groups are cation exchangers.

Size exclusion chromatography:

 Separation by size based on differential exclusion pores within the

solid stationary phase, smaller particles have larger retention time
compared to large particles when they pass through a porous matrix
  The total bed volume (Figure 5A.4) of the packed chromatography
column, Vt, is equal to the volume outside the porous beads (Vo)
plus the volume inside the beads (Vi) plus the volume actually
occupied by the bead material (Vg): Vt Vo Vi Vg. (Vg is typically
less than 1% of Vt and can be conveniently ignored in most
applications.)

Hydrophobic Interaction Chromatography

 exploits the hydrophobic nature of proteins in purifying them

 Proteins are passed over a chromatographic column packed with a
support matrix to which hydrophobic groups are covalently linked
 . In the presence of high salt concentrations, proteins bind to the
phenyl groups by virtue of hydrophobic interactions. Proteins in a
mixture can be differentially eluted from the phenyl groups by
lowering the salt concentration

Affinity Chromatography

 Affinity purification strategies for proteins exploit the biological

function of the target protein
 Proteins function in binding with specific molecules, these
molecules can be permanently bound to an insoluble matrix, , then
the protein of interest, in displaying affinity for its ligand, becomes
bound and immobilized itself
, the protein is dissociated or eluted from the matrix by the addition
of high concentrations of the free ligand in solution

Proteins classified by shape and solubility:

 Fibrous: linear proteins that function in structural role, are

hydrophobic
 Globular: rough spherical shape that has hydrophilic amino acid
side chain facing exterior, making it hydrophilic
 Membrane: proteins embedded in the membrane, hydrophobic
amino acid side chain facing exterior, allowing it to associate with
the lipid bilayer

Primary structure: the amino acid sequence

Secondary structure: the hydrogen-bonding interactions between adjacent

amino acid resides
Tertiary structure: When the polypeptide chains of protein molecules bend
and fold in order to assume a more compact three-dimensional shape

Quaternary structure: Many proteins consist of two or more interacting

polypeptide chains of characteristic tertiary structure

Differences between primary structure and the rest of the structures:

Whereas the primary structure of a protein is determined by the
covalently linked amino acid residues in the polypeptide backbone,
secondary and higher orders of structure are determined principally by
noncovalent forces such as hydrogen bonds and ionic, van der Waals, and
hydrophobic interactions.

Conformation vs configuration:

 Conformation describes the various spatial arrangements of a

molecule that can be interconverted through rotation around single
bonds without breaking any chemical bonds
 Configuration refers to the arrangement of atoms around a specific
bond or group of bonds, which cannot be changed without breaking
those bonds

How Is the Amino Acid Analysis of Proteins Performed:

 Acid Hydrolysis Liberates the Amino Acids of a Protein: peptide

bonds are hydrolysed by strong acid or strong base , acid hydrolysis
it proceeds without racemization and with less destruction of certain
amino acids
 Chromatographic methods

What are the complication with amino acid analysis: Amino acid
analysis itself does not directly give the number of residues of each
amino acid in a polypeptide, but if the molecular weight and the exact
amount of the protein analyzed are known (or the number of amino
acid residues per molecule is known), the molar ratios of amino acids in
the protein can be calculated. Amino acid analysis provides no
information on the order or sequence of amino acid residues in the
polypeptide chain

Terminal ends in polypeptide chain: has only two ends, an amino-

terminal, or N-terminal, end and a carboxy-terminal, or C-terminal, end
 How amino acids are read: read from the N-terminal end of the
polypeptide chain through to the C-terminal end

What was the significance of sangers findings: Sanger’s results clearly

established that all of the molecules of a given protein have a fixed
amino acid composition, , proteins are well defined chemically

What is the proteome: the entire set of proteins encoded by a genome

Protein sequence determination – 6 steps:

1.) Separate polypeptide chains :

2.) Eliminate disulfide bonds
3.) Identify N-terminal and C-terminal amino acid residues
4.) Cleave polypeptide chain at specific sites chemically or
enzymatically and determine the amino acid sequence of each
fragment
5.) Repeat step 3 with a different cleavage procedure in order to
obtain a different, overlapping set of peptide fragments
6.) Reconstruct the protein amino acid sequence from the
sequences of overlapping fragments

Heteromultimer: more than one type of polypeptide chain

Separation of polypeptide chain : , most multimeric proteins can be

dissociated by exposure to pH extremes, 8 M urea, 6 M guanidinium
hydrochloride, or high salt concentrations. All of these treatments disrupt
polar interactions such as hydrogen bonds both within the protein
molecule and between the protein and the aqueous solvent

Cleavage of disulfide bridge:

 Oxidation of a disulfide by performic acid results in the formation of

two equivalents of cysteic acid, Because these cysteic acid side
chains are ionized SO3 groups, electrostatic repulsion, prevents SOS
recombination.
 Alternatively, sulfhydryl compounds such as 2-mercaptoethanol or
dithiothreitol (DTT) readily reduce SOS bridges to regenerate two
cysteineOSH side chain, To prevent this, SOS reduction must be
followed by treatment with alkylating agents such as iodoacetate or
3-bromopropylamine, which modify the SH groups and block
disulfide bridge formation
N-terminal analysis via ednam degradation:

 Identifies the amino acid at the N-terminus of a protein and can

sequentially determine subsequent residues.
 Uses phenylisothiocyanate (Edman reagent) to label and cleave
the N-terminal amino acid under weakly basic conditions.
 The cleaved residue is converted to a PTH derivative, identifiable
via chromatography.

C-terminal analysis using carboxypeptidases:

 Identifies the C-terminal amino acid of polypeptides through

enzymatic cleavage
 Successively removes amino acids from the C-terminus
 Enzyme used : Carboxypeptidase

Steps 4 and 5. Fragmentation of the Polypeptide Chain:

 Produce manageable peptide fragments for sequencing via end-

group analysis
 A. Trypsin Specificity: Cleaves C-terminal side of Lys (K) and
Arg (R). Advantage: Highly specific, ideal for first-pass
digestion in sequencing.
 B. Chymotrypsin Specificity: Prefers Phe (F), Tyr (Y), Trp (W) at
the cleavage site.
 Strategy for Protein Sequencing:
Digest protein separately with trypsin and chymotrypsin.
Sequence the fragments (e.g., via Edman degradation).
Overlap the fragments to deduce the full protein sequence.

 Trypsin = Highly specific, predictable fragments.

Chymotrypsin = Broader specificity, useful for overlapping

peptide mapping.

Reconstruction of the Overall Amino Acid Sequence:

Peptides generated from specific fragmentation of the polypeptide can be

aligned to reveal the overall amino acid sequence
What are homologous proteins: Proteins sharing a significant degree of
sequence similarity and structural resemblance are said to be
homologous, proteins that perform similar function are also referred to as
homologous.

Type of homologous proteins: orthologous and paralogous

Orthologous proteins function and origin:

 proteins are proteins from different species that have homologous

amino acid sequences
 arose from a common ancestral gene during evolution

Paralogous proteins function and origin:

 proteins are proteins found within a single species that have

homologous amino acid sequences
 paralogous proteins arose through gene duplication

what suggest common ancestry for proteins: proteins with related

functions often show a high degree of sequence similarity

what causes deviations; chance mutations change the amino acid

sequence, causing new protein

molecular recognition via ligand binding: All protein functions depend

on specific, reversible, noncovalent interactions with target molecules
(ligands)

Ligand-Binding Features

 Specificity: Arises from a complementary binding site shaped by:

o Amino acid side chains.

o Charge distribution.

o Hydrogen-bond donors/acceptors.

 Induced Fit: Binding often triggers conformational changes in the

protein, enhancing ligand affinity.
What determines The ability of a particular protein to carry out its function
in nature:

is normally determined by its overall three-dimensional shape, or

conformation

what principles link the three-dimensional structure of proteins and their

biological function:

 Function depends on structure.

 Structure depends both on amino acid sequence and on weak,
noncovalent forces.
 The number of protein folding patterns is very large but finite.
 The structures of globular proteins are marginally stable.
 Marginal stability facilitates motion.
 Motion enables function.

Significance of hydrogen bond in protein structures: Hydrogen bonds (H

bonds) play a crucial role in stabilizing protein structures.

Location of hydrogen bonds in proteins and their significance:

 Side chains capable of H-bonding are usually on the protein surface,

bonding with water or other surface residues.

 H bonds in the protein interior (away from water) provide significant

stabilization energy

Significance of hydrophobic interactions in protein structures: Hydrophobic

interactions are a key driving force in protein folding and stability.

Why do Nonpolar (hydrophobic) side chains cluster together:

 Nonpolar (hydrophobic) side chains cluster together to avoid contact

with water, minimizing unfavorable interactions with the polar
solvent.

 These interactions are entropically driven, as releasing ordered

water molecules increases system entropy.

Hydrophobic interactions role in protein folding:

 Hydrophobic clustering is the main driving force behind protein

folding.
  The protein core is predominantly composed of hydrophobic
residues, shielding them from water.

Significance ionic interactions in proteins :

Ionic (electrostatic) interactions play a significant role in protein stability

and function.

Nature of ionic interactions:

 Occur between oppositely charged groups (attraction) or like

charges (repulsion).

 Charged amino acids (e.g., Lys⁺, Arg⁺, His⁺, Asp⁻, Glu⁻) and
terminal residues (N-term⁺, C-term⁻) participate in these
interactions

Location of charged groups in amino acids:

 Charged residues are typically found on the protein surface, where

they interact favorably with water.
 Placing charged residues in the hydrophobic core is energetically
unfavorable.

Influence of salts on charged proteins:

 Dissolved salts (e.g., NaCl) can weaken ionic interactions by

competing for charged sites.

 High salt concentrations may screen electrostatic attractions,

reducing their stabilizing effect.

Significance of Van der Waals (vdW) interactions in protein stability:

Van der Waals (vdW) interactions are weak but collectively significant
forces in protein structure.

Nature of vdW interactions:

 Include attractive (instantaneous dipole-induced dipole)

and repulsive forces between adjacent nonbonded atoms.
  Arise from fluctuations in electron charge distributions.

Strength and cumalitve effect of vdW in protein stability:

 Individually weak (0.4–4.0 kJ/mol), but their large number in proteins

makes them a major stabilizing factor.

 Tightly packed protein interiors (e.g., in ribonuclease A, lysozyme,

cytochrome c, myoglobin) rely heavily on vdW forces for stability

Role of vdw in protein stability:

 Dominant in the hydrophobic core, where dense atomic packing

maximizes these interactions.

 Contribute significantly to the overall free energy stabilization of

folded proteins.

How, then, does nature dictate the manner of protein folding to generate
the three-dimensional structure that optimizes and balances these many
forces: All of the information necessary for folding the peptide chain into
its “native” structure is contained in the amino acid sequence of the
peptide

What is the Peptide Bond Rigidity & Rotational Freedom:

 The peptide bond's planarity restricts backbone flexibility, leaving

only two rotational degrees of freedom per residue:

o φ (phi): Rotation around the N–Cα bond.

o ψ (psi): Rotation around the Cα–C=O bond.

 Each α-carbon connects two peptide planes, defining the

backbone's 3D path.

Steric Constraints on φ and ψ Angles:

 Certain (φ, ψ) combinations are sterically forbidden:

o φ ≈ 0°, ψ ≈ 180°: Causes carbonyl oxygen clashes.

o φ ≈ 180°, ψ ≈ 0°: Leads to H-atom overlaps (N–H

repulsion).

 Steric clashes limit allowed conformations, influencing protein

folding.
Ramachandran Plot: Mapping Allowed Conformations:

 Most (φ, ψ) combinations are sterically prohibited (empty regions).

 Clusters correspond to common secondary structures (e.g., α-
helices, β-sheets).

How are local polypeptide conformations stabilised:

Local polypeptide conformations stabilized by hydrogen bonds between

the amide proton (N–H) of one peptide group and the carbonyl oxygen
(C=O) of another.

Two major types of polypeptide stabilisation forms:

 Two Major Types: α-helices and β-pleated sheets

Characteristics of a-helices:

 Residues per Turn: 3.6 amino acids, with a translation

distance (pitch) of 5.4 Å per turn (1.5 Å/residue).

 Hydrogen Bonding: Each C=O bonds to N–H four residues away,

forming parallel H-bonds along the helix axis.

 Dipole Moment: Aligned peptide bonds create a helix dipole—N-

terminus (partial +), C-terminus (partial –).

o Functional Impact: Negatively charged ligands (e.g.,

phosphates) often bind near the N-terminus.

 Dimensions: ~6 Å diameter (excluding side chains, which project

outward to avoid steric clashes)

 φ ≈ –60°, ψ ≈ –45° to –50° (allowed Ramachandran plot values).

β-Pleated Sheets (β-Structures) Basic Structure: Formed by hydrogen

bonds between adjacent peptide strands, creating a zigzag ("pleated")
backbone with α-carbons at the folds.

Two Types:

 Parallel β-Sheet: Adjacent strands run in the same direction; H-

bonds are bent and less linear.
  Antiparallel β-Sheet: Adjacent strands run in opposite
directions; H-bonds are straighter and stronger.

Differences between parallel and anti-parallel:

 Residue Spacing: 0.347 nm (antiparallel) vs. 0.325 nm (parallel).

 Regularity: Parallel sheets are more uniform in (φ, ψ) angles (φ ≈

–120°, ψ ≈ +105°) compared to antiparallel sheets (φ ≈ –135°, ψ ≈
+140°).

 Size: Parallel sheets usually have ≥5 strands; antiparallel sheets

can be as small as 2 strands.

 Side-Chain Distribution:

o Parallel: Hydrophobic residues on both sides of the sheet.

o Antiparallel: Hydrophobic residues on one side, requiring

alternating hydrophilic/hydrophobic residues in the sequence.

B-turns role in globular proteins:

 Enable chain reversal to form compact, globular structures.

 Stabilized by an H-bond between the C=O of residue i and N–H of

residue i+3.

Types of b-turns:

 Type I: Most common; proline often occupies position 3.

 Type II: Proline prefers position 2; position 3 favors glycine (due

to steric constraints).

The tertiary structure of a protein describes the complete three-

dimensional arrangement of its polypeptide chain, stabilized by
interactions between amino acid side chains. The process of protein
folding is dictated by the primary structure—the sequence of amino
acids—which contains all the necessary information for the protein to
adopt its native conformation. While chaperone proteins assist in
folding within cells, experiments have shown that many proteins can
spontaneously refold in dilute solutions without external help.

The first high-resolution protein structures—myoglobin (determined

by John Kendrew) and hemoglobin (by Max Perutz)—were solved in
the late 1950s after decades of work. These breakthroughs laid the
foundation for understanding protein folding principles, which include:

1. Secondary Structure Formation:

 o α-helices and β-sheets arise from extensive hydrogen
bonding along the polypeptide backbone.

o These structures are energetically favorable and form

whenever possible.

2. Packing of Secondary Structures:

o Proteins are not stable as single-layer structures; instead, α-

helices and β-sheets pack tightly together in specific
arrangements.

o Common packing motifs include α-α bundles, β-

sandwiches, and α/β barrels.

3. Loop Regions Are Short and Direct:

o The segments connecting secondary structures (loops and

turns) are typically short and avoid complicated knots or
twists.

o This ensures efficient folding and minimizes entropic

penalties.

4. Maximizing Stability:

o Proteins fold into their most thermodynamically

stable conformation.

o Stability is achieved through:

 Hydrogen bonding (both within secondary structures

and between side chains).

 Minimizing solvent-exposed hydrophobic

residues (the hydrophobic effect).

The Hydrophobic Effect Drives Folding

Proteins contain a mix of hydrophobic (water-repelling)

and hydrophilic (water-attracting) residues. If a protein were entirely
hydrophilic, it would remain unfolded, as all side chains could interact
favorably with water. However, hydrophobic residues disrupt water’s
hydrogen-bonding network, increasing free energy. To minimize
this, hydrophobic residues cluster in the protein’s core, shielding
them from water and driving compaction.

A challenge in this model is that the peptide backbone—despite being

polar—must also enter the hydrophobic core. To compensate, backbone
NH and C=O groups form hydrogen bonds (e.g., in α-helices and β-
sheets), ensuring stability even in the protein’s interior.

Conclusion

Protein tertiary structure is a result of hydrogen bonding, hydrophobic

packing, and thermodynamic stability. The hydrophobic effect forces
folding, while secondary structures provide stability. Understanding these
principles helps explain how proteins achieve their functional shapes and
how misfolding can lead to disease.

Fibrous Proteins: Structure and Function

Overview of Fibrous Proteins

Fibrous proteins are a class of proteins characterized by elongated,

parallel polypeptide chains that form strong, insoluble fibers or
sheets. Unlike globular proteins, which are compact and soluble, fibrous
proteins are mechanically robust and often serve structural roles in
organisms.

α-Keratin: A Coiled-Coil Helical Protein

 Found in: Hair, nails, claws, horns, and wool.

 Structure:

o Composed of central α-helical rod domains (311–314

residues) flanked by nonhelical regions.

o Two right-handed α-helices twist together to form a left-

handed coiled coil.

o The pitch (0.51 nm vs. 0.54 nm in standard α-

helices) indicates a slight helical tilt, optimizing stability.

 Sequence Features:

o Quasi-repeating 7-residue segments (a-b-c-d-e-f-g)ₙ,

where positions a and d are usually hydrophobic (e.g., Leu,
Ile, Val).

o These nonpolar residues form a hydrophobic stripe along

the helix, driving dimerization to shield them from water.

 Stabilization:

o Hydrophobic packing between helices enhances stability.

 o Disulfide bonds (in harder keratins, like hair and nails)
increase rigidity by cross-linking cysteine residues.

 Biological & Cosmetic Relevance:

o Permanent waves ("perms") involve breaking and

reforming disulfides to restructure curls.

o Temporary curls ("sets") rely on hydrogen bond

rearrangement from wetting/drying, which can be disrupted
by humidity ("frizz").

Fibroin (Silk) & β-Keratin: β-Sheet Proteins

 Found in: Silkworm cocoons (Bombyx mori), spider silk, and bird
feathers.

 Structure:

o Stacked antiparallel β-sheets with alternating small (Gly)

and bulky (Ala, Ser) residues.

o Glycine residues align on one side,

while alanine/serine residues face the opposite side,
allowing tight packing.

 Properties:

o High tensile strength due to extended β-sheet

conformation.

o Flexibility from weak van der Waals forces between sheets.

Key Differences Between α-Keratin and β-Keratin/Fibroin

Feature α-Keratin Fibroin / β-Keratin

Secondary
α-Helices (coiled coil) Antiparallel β-Sheets
Structure

Hydrophobic packing, Hydrogen bonds, tight

Stabilization
disulfides Gly/Ala packing

Moderate (disulfides High (due to weak inter-

Flexibility
increase rigidity) sheet forces)

Silk, spiderwebs, bird

Examples Hair, nails, horns
feathers

Conclusion
Fibrous proteins like α-keratin and fibroin/β-keratin exemplify
how secondary structure dictates mechanical function. α-
Keratin’s coiled-coil helices provide strength and elasticity, while
silk’s β-sheets offer exceptional toughness. These proteins highlight the
evolutionary optimization of sequence, structure, and chemical
interactions for specialized biological roles.

The Coiled-Coil Motif in Protein Structure

Discovery and Significance

 First identified in 1953 by Linus Pauling, Robert Corey, and

Francis Crick in fibrous proteins like keratin and myosin.

 A common structural motif found in many proteins, where two,

three, or four α-helices twist together into a superhelix.

Structure and Assembly

 Helical Arrangement:

o Can be parallel or antiparallel.

o Typically involves 2-4 α-helices wound into a left-handed

supercoil.

 Key Feature: Heptad Repeat (7-residue pattern)

o A quasi-repeating sequence (a-b-c-d-e-f-g)ₙ, where

positions a and d are usually hydrophobic.

o The 3.5 residues per turn (instead of 3.6 in standard α-

helices) ensures hydrophobic side chains align every two
turns, enabling tight packing.

Why the Heptad Repeat?

 A standard α-helix (3.6 residues/turn) would shift side-chain

positions, preventing consistent packing.

 A slight left-handed twist reduces it to 3.5 residues/turn,

making side chains repeat every 7 residues (2 turns).

 This allows hydrophobic core formation, stabilizing the coiled

coil.

Examples of Coiled-Coil Proteins

  Keratin (hair, nails), myosin (muscle fibers), transcription
factors (e.g., leucine zippers).

 Used in both structural and regulatory roles.

Conclusion

The coiled-coil motif is a versatile, stable structure stabilized

by hydrophobic packing and a heptad repeat sequence. Its discovery
was pivotal in understanding how α-helices assemble into higher-
order structures, influencing fields from cell biology to materials
science.

Collagen – The Triple Helix of Connective Tissues

Biological Importance

Collagen is a rigid, fibrous protein that serves as the primary

structural component of connective tissues, including:

 Tendons, cartilage, bones, teeth, skin, and blood vessels

 Provides high tensile strength, enabling mechanical stress

resistance (e.g., running, jumping)

 Injuries like bone fractures and tendon tears often involve

collagen matrix damage

Molecular Structure

 Basic Unit: Tropocollagen

o Composed of three intertwined polypeptide

chains (~1000 residues each)

o Forms a right-handed triple helix (300 nm long, 1.4 nm

diameter)

o Each chain adopts a left-handed helical

conformation (unlike α-helices)

 Types of Collagen

o Type I (most common): Two α1(I) chains + one α2(I) chain

(found in skin, bones, tendons)

o Type II: Three identical chains (cartilage)

o Type III: Three identical chains (blood vessels)

Unique Amino Acid Composition

 Glycine (Gly): Every third residue (required for tight packing in

the helix core)

 Proline (Pro) & Hydroxyproline (Hyp): Make up ~30% of

residues

 Modified Residues:

o 4-Hydroxyproline, 3-Hydroxyproline, 5-
Hydroxylysine (post-translational modifications)

o Synthesis requires:

 Prolyl hydroxylase & lysyl hydroxylase

 Vitamin C (ascorbic acid), O₂, Fe²⁺, α-

ketoglutarate

Triple Helix Formation

 Sequence Motif: Repeating Gly-X-Y (X = often Pro, Y = often

Pro/Hyp)

 Structural Features:

o Extended conformation (2.9 Å rise per residue vs. 1.5 Å in

α-helices)

o 3.3 residues per turn

o Gly faces the crowded center; X and Y residues stabilize

via:

 Interchain H-bonds (Gly-NH to X-CO)

 Hydroxyproline-mediated H-bonding

 Fibril Assembly:

o Tropocollagen molecules form staggered arrays (68 nm

repeat pattern in EM)

o 40-nm "hole zones" between molecules:

 Glycosylation sites (carbohydrates attached to

hydroxylysine)

 Nucleation sites for bone

mineralization (hydroxyapatite crystal formation)

Functional Implications
  Mechanical Stability: Tight Gly-X-Y packing and H-bonding prevent
stretching

 Bone Formation: Hole zones guide hydroxyapatite

deposition in bones

 Disease Links:

o Scurvy (vitamin C deficiency → impaired Hyp synthesis →

weak collagen)

o Osteogenesis imperfecta (brittle bone disease from Gly

mutations)

Conclusion

Collagen’s triple-helix structure—enabled by its Gly-Pro-Hyp-rich

sequence and post-translational modifications—is essential for tissue
integrity. Its staggered fibril arrangement not only provides strength
but also directs bone mineralization, highlighting its dual role
in mechanical support and biomineralization.

Protein Domains – Nature’s Modular Building Blocks

Key Concepts:

1. Domain Definition & Structure:

o Domains are compact, independently folded units within

larger proteins, typically stable in aqueous solution.

o Most domains consist of 100–250 amino acids, forming

globular structures with:

 Hydrophobic cores (stabilizing the fold).

 Hydrophilic surfaces (ensuring solubility).

o Larger proteins (>250 residues) often consist of multiple

domains connected by flexible linker regions (e.g., TonEBP, a
two-domain DNA-binding protein).

2. Functional Modularity:

o Each domain often performs a specific function (e.g., ligand

binding, catalysis, protein-protein interactions).

o In multidomain proteins, the combined functions of domains

enable complex biological roles.
 o Example: Immunoglobulins (antibodies) use distinct domains
for antigen binding and immune signaling.

3. Evolutionary Significance:

o Domains are evolutionary units—many proteins evolved

via gene duplication and fusion of ancestral domain-coding
sequences.

o ~90% of eukaryotic domains have been duplicated,

allowing functional diversification.

o Some proteins contain repeated

domains (e.g., fibronectin with multiple type-III domains).

4. Structural Diversity:

o Common domains (e.g., SH3, immunoglobulin fold,

Rossmann fold) recur across proteins (see Figure 6.25).

o Discontinuous domains: Rarely, a domain’s sequence is

interrupted by another structural region (Figure 6.24).

Implications:

 Protein Engineering: Domains can be "mixed and matched" to

design synthetic proteins.

 Disease Mechanisms: Mutations in critical domains (e.g., kinase

domains in cancer) disrupt function.

 Evolutionary Efficiency: Reusing stable domains accelerates the

development of new functions.

Conclusion:

Protein domains are nature’s modular toolkit, enabling structural

stability, functional versatility, and evolutionary innovation. Their
combinatorial assembly explains the diversity of protein functions in
biology

Intrinsically Unstructured Proteins (IUPs) – Functional Flexibility

in Disorder

Key Characteristics of IUPs

 Lack stable 3D structure: Exist in a dynamic, extended

conformation with high flexibility.
  Critical for cellular functions: Despite being unfolded, they play
essential roles (e.g., signaling, transcription).

 Bind targets over large surfaces:

Example: p27 binds Cdk2/cyclin A across its entire length,
interacting with multiple domains (Figure 6.40).

Sequence Features Predict Disorder

 High net charge (enriched in E, K, R, G, Q, S, P).

 Low hydrophobicity (depleted in I, L, V, W, F, Y, C, N).

 Bioinformatics tools can predict disordered regions with >80%

accuracy.

Genomic and Functional Insights

 Prevalence increases with organism complexity:

o 2% in archaea → 4.2% in bacteria → 33% in eukaryotes.

 Two types of disorder:

1. Fully disordered proteins (e.g., transcriptional

coactivators).

2. Local disordered segments (30–40 residues) within folded

proteins.

Biological Advantages of Disorder

1. Multifunctional adaptability:

o Bind diverse ligands (proteins, DNA) via conformational

plasticity.

o Example: β-catenin binds disordered TCF3 to regulate

transcription (Figure 6.40).

2. Efficient interaction interfaces:

o Disordered regions create larger binding surfaces than folded

proteins of similar size.

o Reduces need for larger proteins, minimizing cellular crowding

and genome size.

Evolutionary Implications

 Disorder enables functional versatility in complex organisms

(e.g., signaling hubs in eukaryotes).
  May drive efficiency in protein interaction networks by reducing
structural constraints.

Conclusion

IUPs defy the traditional "structure-function" paradigm, demonstrating

that disorder itself is functional. Their flexibility supports diverse
interactions, optimizing cellular processes while conserving genomic and
spatial resources. This discovery reshapes our understanding of protein
evolution, particularly in higher organisms.

Protein Oligomerization and Quaternary Structure

1. Definition and Prevalence of Oligomeric Proteins

 Oligomers are complexes of two or more protein subunits

(monomers) held together by noncovalent interactions.

 Common in nature, with examples including:

o Homomultimers: Identical subunits (e.g., liver alcohol

dehydrogenase, glycogen phosphorylase).

o Heteromultimers: Different subunits (e.g., hemoglobin =

α₂β₂).

o Large polymeric assemblies: Ribosomes, actin-myosin

filaments, microtubules (tubulin polymers).

2. Types of Subunit Interactions

 Isologous associations: Symmetric binding between identical

interfaces (e.g., dimers with twofold symmetry like transthyretin
tetramers).

 Heterologous associations: Asymmetric binding between

complementary, nonidentical interfaces (e.g., tubulin αβ-dimers
polymerizing into microtubules).

 Open vs. Closed Structures:

o Closed: Finite assemblies (e.g., hemoglobin).

o Open: Indefinite polymerization (e.g., actin filaments, viral

capsids like HIV).

3. Functional Advantages of Oligomerization

  Stability:

o Reduces surface-to-volume ratio, shielding hydrophobic

residues.

o Minimizes solvent exposure, lowering free energy.

 Genetic Economy:

o Encoding a self-assembling monomer is more efficient than a

single large polypeptide (e.g., HIV protease vs. cellular
proteases).

 Catalytic Efficiency:

o Active sites often form at subunit interfaces (e.g., glutamine

synthetase).

o Subunits can perform sequential reactions (e.g., tryptophan

synthase’s αβ-subunits pass intermediates directly).

 Regulation via Cooperativity:

o Allostery: Ligand binding at one subunit alters affinity at

others (e.g., O₂ binding to hemoglobin).

o Enables precise control of metabolic pathways (see Chapter

15).

4. Biological Implications

 Structural Roles: Tubulin → microtubules; actin → muscle

contraction.

 Disease Relevance: HIV coat proteins form capsids; mutations

disrupting subunit interactions cause dysfunction (e.g.,
hemoglobinopathies).

 Evolutionary Efficiency: Modular assembly allows functional

complexity with minimal genetic material.

Conclusion

Quaternary structure—through symmetry, stability, and cooperative

regulation—expands protein functionality beyond monomeric limits.
From metabolic enzymes to viral particles, oligomerization is a
cornerstone of biological complexity, balancing genetic economy with
sophisticated control mechanisms.

Extra notes:
post-Translational Modifications (PTMs) of Amino Acid
Side Chains in Proteins:

Phosphorylation

 Definition: Addition of a phosphate group (PO₄³⁻) to

an amino acid side chain, typically by kinases.
 Role: Regulates enzyme activity, signal transduction,
and protein-protein interactions.

Acetylation

 Definition: Addition of an acetyl group (CH₃CO-) to an

amino acid side chain.
 Role: Modifies protein function, chromatin structure
(histone acetylation), and metabolic regulation.

The Anfinsen Experiment: Rationale and Significance

1. Denaturation: RNase A was treated with urea (chaotropic
agent) and β-mercaptoethanol (reducing agent), disrupting:
o Non-covalent interactions (e.g., hydrogen bonds,
hydrophobic packing).
o Disulfide bonds (by reducing cysteine residues to -SH
groups).
→ The protein became unfolded and inactive.
2. Renaturation:
o The denaturants were slowly removed, allowing the
protein to refold.
o Oxygen in the buffer re-formed the correct disulfide
bonds (oxidizing -SH back to -S-S-).
→ RNase A regained its enzymatic activity, proving it
folded back to its native structure.
3. Control Experiment:
o If the protein was re-oxidized without removing urea
first, it formed incorrect disulfide bonds (scrambled
structures) and remained inactive.
→ This showed that non-covalent interactions must
guide disulfide bond formation during folding.

What do enzymes allow cells to do : accelerate and control the rates of

vitally important biochemical reactions
What are regulatory enzyme: capable of sensing the momentary
metabolic needs of the cell and adjusting their catalytic rates accordingly

3 distinctive features of enzymes :

catalytic power, specificity, and regulation

Catalytic Power: the Ratio of the Enzyme-Catalyzed Rate of a Reaction to

the Uncatalyzed Rate

Specificity: the Term Used to Define the Selectivity of Enzymes for Their
Substrates

What are substrates: the Term Used to Define the Selectivity of Enzymes
for Their Substrates

What is the active site: The specific site on the enzyme where substrate
binds and catalysis occurs

Regulation: Regulation of Enzyme Activity Ensures That the Rate of

Metabolic Reactions Is Appropriate to Cellular Requirements

What are coenzymes and cofactors: Coenzymes and Cofactors Are

Nonprotein Components Essential to Enzyme Activity

What are prosthetic groups: a coenzyme is firmly associated with its

enzyme, perhaps even by covalent bonds, and it is difficult to separate the
two. Such tightly bound coenzymes are referred to as prosthetic groups

Holoenzyme vs apoenzyme:

 The catalytically active complex of protein and prosthetic group is

called the holoenzyme.
 The protein without the prosthetic group is called the apoenzyme; it
is catalytically inactive

Aim of enzyme kinetics: In enzyme kinetics, we seek to determine the

maximum reaction velocity that the enzyme can attain and its binding
affinities for substrates and inhibitors.

When is irreversibility assumed in chemical kinetics: Irreversibility is easily

assumed if the rate of P conversion to A is very slow or the concentration
of P (expressed as [P]) is negligible under the condition chosen
What determines The velocity, v, or rate, of the reaction A⎯→P : is the
amount of P formed or the amount of A consumed per unit time, t.

What is the rate law: The mathematical relationship between reaction rate
and concentration of reactant(s) is the rate law

What determines reaction order: For an elementary reaction, the order for
any reactant is given by its exponent in the rate equation.

What is the molecularity of a reaction: The number of molecules that must

simultaneously interact is defined as the molecularity of the reaction

unimolecular reactions: involve a single reactant molecule undergoing a

change to form products, the molecularity equals 1

bimolecular reaction: a chemical reaction where two chemical species

collide and react, forming a product, the molecularity equals 2

Energy diagram components:

Transition state: the state at which reactants have the required energy to
undergo the required chemical reaction

Initial state: The average free energy of A molecules

Final state: The average free energy of P molecules

(Delta)G‡ meaning: activation energy, the energy required to raise the

average energy of 1 mol of reactant (at a given temperature) to the
transition state energy)

Ways in which we increase reaction rate:

Raising temperature: increasing the average energy of reactant molecules

makes the energy difference between the average energy and the
transition-state energy smaller.

Use of catalyst: Catalysts work by lowering the energy of activation rather

than by raising the average energy of the reactants, by combining
transiently with the reactants in a way that promotes their entry into the
reactive, transition-state condition

Characteristics of catalyst in chemical reaction:

(1)They are regenerated after each reaction cycle (A⎯→P), and

therefore can be used over and over again
 (2)catalysts have no effect on the overall free energy change in
the reaction, the free energy difference between A and P

key differences between simple chemical reactions and enzyme-catalyzed

reactions:

1. Simple Reactions (A → P):

o The rate (v) increases linearly with reactant concentration

([A]), following first-order kinetics (slope = rate constant k).

2. Enzyme-Catalyzed Reactions (Single Substrate S → P):

o At low [S], v increases proportionally with [S] (first-order

kinetics).

o At high [S], v plateaus and becomes independent of [S],

approaching a maximum rate (V_max) (zero-
order kinetics).

Why does this plateau occur in enzyme catalysed reactions:

Saturation effect: all enzyme active sites are occupied by substrate,

forming enzyme-substrate complexes.

The Michaelis-Menten equation is the foundational model for enzyme

kinetics, describing how reaction velocity (vv) depends on substrate
concentration ([S][S]). Key points include:

1. Basic Reaction Scheme:

o Enzymes (EE) bind substrates (SS) reversibly to form an

enzyme-substrate complex (ESES), which then breaks down to
product (PP):

E+S⇌k1k−1ES→k2E+PE+Sk−1⇌k1ES→k2E+P

o The steady-state assumption (Briggs & Haldane, 1925)

states that [ES][ES] remains constant during the reaction.

2. Michaelis-Menten Equation:

o The reaction velocity is given by:

v=Vmax[S]Km+[S]v=Km+[S]Vmax[S]

where:

 Vmax=k2[ET]Vmax=k2[ET] (maximal velocity at

saturating [S][S]),
  Km=k−1+k2k1Km=k1k−1+k2 (Michaelis constant,
reflecting enzyme-substrate affinity).

3. Key Interpretations:

o When [S]=Km[S]=Km, v=Vmax2v=2Vmax.

o At low [S][S] ([S]≪Km[S]≪Km), v≈VmaxKm[S]v≈KmVmax

[S] (first-order kinetics).

o At high [S][S] ([S]≫Km[S]≫Km), v≈Vmaxv≈Vmax (zero-order

kinetics).

4. Turnover Number (kcatkcat):

o The number of substrate molecules converted to product per

enzyme per second:

kcat=Vmax[ET]kcat=[ET]Vmax

o Example: Catalase has a kcatkcat of 40 million s−1s−1, while

lysozyme has 0.5 s−1s−1.

5. Catalytic Efficiency (kcat/Kmkcat/Km):

o Measures how efficiently an enzyme converts substrate to

product at low [S][S].

o The upper limit (~108−109 M−1s−1108−109M−1s−1) is set

by diffusion rates. Enzymes approaching this are "catalytically
perfect."

6. Linear Transformations:

o Lineweaver-Burk Plot (1/v1/v vs. 1/[S]1/[S]):

1v=KmVmax⋅1[S]+1Vmaxv1=VmaxKm⋅[S]1+Vmax1

o Hanes-Woolf Plot ([S]/v[S]/v vs. [S][S]):

[S]v=1Vmax[S]+KmVmaxv[S]=Vmax1[S]+VmaxKm

o These plots allow accurate determination

of KmKm and VmaxVmax.

Key Assumptions:

 Only one substrate is varied (others held constant).

 Initial velocities are measured ([P]≈0[P]≈0).

 [S]≫[ET][S]≫[ET], and steady-state conditions apply.

Competitive Inhibition

Effect:

 KmKm increases (apparent KmKm, KmappKmapp)

 VmaxVmax remains unchanged

Reason:

 The inhibitor (I) binds reversibly to the active site, competing

directly with the substrate (S).

 At high [S], the substrate can outcompete the inhibitor,

so VmaxVmax is still achievable.

 However, more substrate is needed to reach half

of VmaxVmax (higher KmKm), because some enzyme molecules
are bound to I instead of S.

Pure Noncompetitive Inhibition

Effect:

 KmKm remains unchanged

 VmaxVmax decreases

Reason:

 The inhibitor (I) binds to a site other than the active site, causing
a conformational change that reduces catalytic efficiency.

 Since I does not interfere with S binding, KmKm (substrate affinity)

stays the same.

 However, some enzyme molecules are permanently

inactivated (either as EI or ESI), reducing the total active enzyme
and thus lowering VmaxVmax.

Mixed Noncompetitive Inhibition

Effect:

 KmKm may increase or decrease

 VmaxVmax decreases

Reason:

 The inhibitor (I) binds to a separate site but affects

both substrate binding and catalysis.
 o If I reduces S binding affinity → KmKm increases.

o If I stabilizes the ES complex → KmKm decreases.

 Regardless, some enzyme molecules are nonfunctional,

so VmaxVmax decreases.

Uncompetitive Inhibition

Effect:

 KmKm decreases

 VmaxVmax decreases

Reason:

 The inhibitor (I) binds only to the ES complex, not free enzyme.

 This stabilizes the ES complex, effectively increasing substrate

affinity (lower KmKm).

 However, the ESI complex is nonproductive, so fewer active

enzymes are available, reducing VmaxVmax.

5. Irreversible Inhibition

Effect:

 Effectively reduces VmaxVmax (like noncompetitive inhibition)

 KmKm may appear unchanged (if inhibition is uniform)

Reason:

 The inhibitor covalently modifies the enzyme, permanently

inactivating it.

 The remaining active enzyme molecules behave normally, but

the total functional enzyme decreases, lowering VmaxVmax.

 Since the binding site is not altered, KmKm remains the same for
the remaining active enzyme.

Summary Table
 Effect
Inhibition Effect Mechanistic
on VmaxVm
Type on KmKm Reason
ax

I competes
Competitive ↑ (Increases) No change
with S at active site

Pure I binds elsewhere,

↓
Noncompetiti No change reducing active
(Decreases)
ve enzyme

Mixed I alters both

↓
Noncompetiti ↑ or ↓ binding and
(Decreases)
ve catalysis

I binds only ES,

Uncompetitiv ↓ stabilizing it but
↓ (Decreases)
e (Decreases) blocking product
formation

No
↓ I permanently
Irreversible change (appare
(Decreases) inactivates enzyme
nt)

Metabolism:

Glycolysis:

What is the glycolysis pathway: Glycolysis is a 10-step metabolic pathway

that converts one glucose molecule into two pyruvate molecules,
producing ATP in the process

Summary of the phases in glycolysis:

1. Energy Investment Phase (5 reactions): Glucose is split into

two glyceraldehyde-3-phosphate molecules, consuming 2 ATP.
 2. Energy Payoff Phase (5 reactions): Each glyceraldehyde-3-phosphate
is converted to pyruvate, generating 4 ATP (net gain of 2 ATP per
glucose).

3. Pyruvate, the end product, is versatile:

1. Under aerobic conditions, it is oxidized to acetyl-CoA for
further breakdown in the TCA cycle.
2. Under anaerobic conditions, it undergoes fermentation (e.g.,
to lactate in muscles or ethanol in yeast).

Energy Efficiency of Glycolysis

Glycolysis converts glucose into lactate (in anaerobic conditions, like in

contracting muscle) with a standard free energy change (ΔG°') of -183.6
kJ/mol:

C6H12O6→2 lactate+2H+(ΔG°′=−183.6 kJ/mol)

 No net oxidation/reduction: Although some steps involve electron

transfers, they balance out, meaning glycolysis is primarily a bond
rearrangement without a net change in electron state.

 ATP production requires energy:

2ADP+2Pi→2ATP+2H2O(ΔG°′=+61 kJ/mol)

Overall coupled reaction:

Glucose+2ADP+2Pi→2lactate+2ATP+2H++2H2O

ΔG°′=−183.6+61=−122.6 kJ/mol

Part one glycolysis

Reaction 1 – Phosphorylation of Glucose by Hexokinase/Glucokinase:

 Enzyme: Hexokinase (most tissues) or glucokinase (liver, pancreas).

 Reaction:

Glucose+ATP→Glucose-6-phosphate+ADP+H+

ΔG°' = -16.7 kJ/mol (highly favorable under standard

conditions, Keq≈850Keq≈850).

Why ATP is Used Early in Glycolysis:

 o Priming Reaction: This step consumes ATP to activate glucose
for downstream energy extraction.

o Thermodynamics:

 ATP hydrolysis provides -30.5 kJ/mol.

 Glucose phosphorylation requires +13.8 kJ/mol.

 Net energy release = -16.7 kJ/mol, driving the reaction

forward.

Why Glucose Phosphorylation is Crucial for Cells

1. Traps Glucose Inside the Cell

o Glucose is neutral and can diffuse out, but glucose-6-

phosphate (G6P) is negatively charged and cannot cross the
membrane.

o Ensures glucose remains inside for metabolic use.

2. Maintains a Concentration Gradient for Uptake

o Rapid conversion to G6P keeps intracellular glucose levels low,

promoting further glucose uptake via facilitated diffusion.

3. Regulatory Control Point

o Since the reaction is highly favourable (far from equilibrium),

it serves as a key control point for glycolysis.

o Enzymes like hexokinase/glucokinase can be regulated to

adjust metabolic flux.

What are kinases: an enzyme that catalyses the transfer of a phosphate

group from ATP to a specified molecule.

Hexokinase Isozymes and Their Roles in Glucose Metabolism

Hexokinase (Types I-III)

o Found in: Most animal tissues (brain, muscle, etc.), plants, and
microbes.

o Key Features:

 Requires Mg²⁺ for activity (true substrate = MgATP²⁻).

  Low Km (0.03–0.3 mM): Highly efficient even at normal
blood glucose levels (~4 mM).

 Allosterically inhibited by glucose-6-phosphate (G6P):

Prevents excessive accumulation when glycolysis slows.

 Broad substrate specificity: Phosphorylates glucose,

mannose, and fructose.

Glucokinase (Hexokinase Type IV)

o Found in: Liver and pancreatic β-cells.

o Key Features:

 High Km (~10 mM): Only active when blood glucose is

high (e.g., after a meal).

 Glucose-specific: Does not act on other hexoses.

 Not inhibited by G6P: Allows liver to continuously

process high glucose loads for storage as glycogen.

Reaction 2 – Isomerization of Glucose-6-Phosphate to Fructose-6-

Phosphate

Key Features of the Reaction

 Enzyme: Phosphoglucoisomerase (PGI, also called phosphoglucose

isomerase).

 Reaction: Converts glucose-6-phosphate (G6P, an

aldose) to fructose-6-phosphate (F6P, a ketose) by shifting the
carbonyl oxygen from C-1 to C-2.

o ΔG°' = +1.67 kJ/mol (near equilibrium, easily reversible).

o Actual cellular ΔG ≈ +2.92 kJ/mol (still close to equilibrium).

 Mechanism: Proceeds via an enediol intermediate, acting on

the open-chain forms of G6P and F6P (despite ring structures
dominating in solution).

 Requires Mg²⁺ for catalytic activity.

Why Is This Step Necessary?

 1. Facilitates Subsequent Phosphorylation:

o The next step (Step 3) phosphorylates C-1, which is easier on

F6P (a primary –OH) than on G6P’s hemiacetal oxygen.

2. Prepares for C-C Bond Cleavage:

o The carbonyl at C-2 in F6P activates C-3, enabling its later

cleavage (in Step 4, aldolase reaction).

Reaction 3 – Phosphofructokinase (PFK) and the Second Priming Reaction

The Reaction

 Enzyme: Phosphofructokinase (PFK) (requires Mg²⁺).

 Process: Phosphorylates fructose-6-phosphate (F6P) at C-1 using ATP

→ fructose-1,6-bisphosphate (F1,6BP).

o ΔG°' = -14.2 kJ/mol (highly favorable, irreversible under

cellular conditions).

o Actual ΔG (erythrocytes) ≈ -18.8 kJ/mol (drives glycolysis

forward).

 Commitment Step:

o Like hexokinase, PFK’s large ΔG makes it a key regulatory

point.

o Once F6P → F1,6BP, glucose is committed to glycolysis (not

storage or alternative pathways).

Regulation of PFK – The "Glycolytic Valve"

1. ATP Inhibition (Allosteric Control):

o ATP binds to a regulatory site (distinct from the active site),

causing cooperative sigmoidal kinetics.

o High [ATP] slows glycolysis (energy-sensing feedback).

o However, [ATP] varies little (~10% drop during exercise),

so AMP is a more sensitive signal.

2. AMP Activation:

o AMP reverses ATP inhibition, sharply increasing when ATP

drops.
 o Adenylate kinase maintains equilibrium:

2ADP⇌ATP+AMP

Tiny ATP decreases cause large AMP increases (amplifying energy demand
signals).

how does amp activate pfk if ATP deactivates it: MP activates

Phosphofructokinase (PFK) even though ATP inhibits it because AMP
directly competes with ATP at the allosteric site of PFK, effectively
reducing the inhibitory effect of ATP

3. Citrate Inhibition (Link to TCA Cycle):

o High [citrate] (from saturated TCA cycle) inhibits PFK,

preventing excess glucose breakdown when energy/
precursors are sufficient.

4. Fructose-2,6-Bisphosphate (F2,6BP) Activation:

o Potent allosteric activator that:

 ↑ PFK’s affinity for F6P.

 Counters ATP inhibition.

o Ensures glycolysis accelerates when glucose is abundant (e.g.,

fed state).

Reaction 4 – Cleavage of Fructose-1,6-Bisphosphate by Aldolase

The Reaction

 Enzyme: Fructose bisphosphate aldolase (Class I in animals, Class II

in bacteria/fungi).

 Process: Cleaves fructose-1,6-bisphosphate (F1,6BP) into two 3-

carbon intermediates:

o Dihydroxyacetone phosphate (DHAP)

o Glyceraldehyde-3-phosphate (G3P)

 Equilibrium:

o Standard ΔG°' = +23.9 kJ/mol (appears unfavorable).

 o Actual cellular ΔG ≈ +0.23 kJ/mol (near equilibrium due to
product dilution)

Reaction 5 – Triose Phosphate Isomerase (TPI) Completes

Glycolysis' First Phase

Key Reaction

 Enzyme: Triose phosphate isomerase (TPI) (a near-"perfect" enzyme

with a turnover rate approaching the diffusion limit).

 Process: Converts dihydroxyacetone phosphate

(DHAP) to glyceraldehyde-3-phosphate (G3P), ensuring both 3-
carbon products of aldolase enter glycolysis’ energy-yielding phase.

 Mechanism: Proceeds via an enediol intermediate, allowing

reversible interconversion of DHAP and G3P.

Energetics & Equilibrium

 Standard ΔG°' ≈ +7.5 kJ/mol (slightly unfavorable for G3P → DHAP).

 Actual cellular ΔG ≈ ~0 (due to rapid consumption of G3P, pulling

equilibrium forward).

 Overall First Phase (5 Steps):

o Net ΔG°' = -2.2 kJ/mol (near equilibrium under standard

conditions).

o Cellular ΔG = -53.4 kJ/mol (highly favorable due to ATP

priming and product removal).

Biological Significance of reaction 5

1. Carbon Symmetry: Makes C1–C3 of glucose equivalent to C6–C4 (via

G3P).

2. Doubles Glycolytic Yield: One glucose → two G3P molecules,

doubling ATP production potential.

3. Catalytic Perfection: TPI’s extreme efficiency ensures no metabolic

bottleneck.

*Part two of glycolysis:

Reaction 6 – Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)
Generates a High-Energy Intermediate

Key Reaction

 Enzyme: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

 Process: Oxidizes glyceraldehyde-3-phosphate (G3P) to 1,3-

bisphosphoglycerate (1,3-BPG) while reducing NAD⁺ to NADH.

o ΔG°' = +6.30 kJ/mol (slightly endergonic under standard

conditions).

o Cellular ΔG ≈ ~0 (driven forward by rapid product utilization).

Mechanism Highlights

1. Covalent Catalysis:

o A cysteine thiol (-SH) on GAPDH attacks G3P’s carbonyl carbon

→ forms a hemithioacetal intermediate.

2. Oxidation & Energy Capture:

o The hemithioacetal is oxidized (via hydride transfer to NAD⁺),

yielding a high-energy thioester intermediate.

3. Phosphate Incorporation:

o Inorganic phosphate (Pᵢ) attacks the thioester → forms 1,3-

BPG (a carboxyl-phosphate mixed anhydride with high
phosphoryl transfer potential).

Energetics & Significance

 1,3-BPG is a high-energy intermediate: Its hydrolysis (ΔG°' = -49.3

kJ/mol) fuels ATP synthesis in the next step (Reaction 7).

 NADH production: Provides reducing power for oxidative

phosphorylation (or fermentation).

 Regulation: Inhibited by iodoacetate (blocks the essential cysteine).

Reaction 7 – Phosphoglycerate Kinase (PGK) and the "Break-Even" ATP

Payoff

Key Reaction
  Enzyme: Phosphoglycerate kinase (PGK) (requires Mg²⁺, acts
on MgADP).

 Process: Transfers a phosphoryl group from 1,3-bisphosphoglycerate

(1,3-BPG) to ADP → 3-phosphoglycerate (3-PG) + ATP.

o Standard ΔG°' = -18.8 kJ/mol (highly favorable).

o Cellular ΔG ≈ 0 kJ/mol (operates near equilibrium in

erythrocytes).

Energetic Significance

 "Breaks Even" for ATP:

o 2 ATP were spent in Phase 1 (priming).

o 2 ATP are produced here (one per 1,3-BPG, so 2 per glucose).

o Net ATP at this stage: 0 (but sets up for 4 ATP total in later
steps).

 Drives Prior Reactions:

o The exergonic PGK reaction pulls forward GAPDH (Reaction

6), aldolase, and triose phosphate isomerase (despite their
slightly unfavorable ΔG°').

Coupling with Reaction 6 (GAPDH)

 Net Coupled Process:

G3P+ADP+Pi+NAD+→3-PG+ATP+NADH+H+

o ΔG°' = -12.6 kJ/mol (spontaneous overall).

 Substrate-Level Phosphorylation:

o Direct ATP synthesis from a high-energy intermediate (1,3-

BPG), unlike oxidative phosphorylation (which uses electron
transport).

Substrate-level phosphorylation: a metabolic reaction where ATP or GTP is

produced directly from the transfer of a phosphate group from a high-
energy intermediate to ADP

Reaction 8 – Phosphoglycerate Mutase Rearranges the Phosphate Group

Key Reaction
  Enzyme: Phosphoglycerate mutase (PGM) (a mutase relocates
functional groups).

 Process: Shifts the phosphoryl group of 3-phosphoglycerate (3-

PG) from C-3 to C-2, yielding 2-phosphoglycerate (2-PG).

o ΔG°' ≈ 0 kJ/mol (near equilibrium; cellular ΔG ≈ +0.83

kJ/mol in erythrocytes).

Mechanism (Yeast/Muscle Enzyme)

1. Phospho-Enzyme Intermediate:

o PGM has a phosphorylated histidine (His-P) at its active site.

2. Phosphoryl Transfer:

o His-P donates its phosphate to C-2 of 3-PG, forming a

transient 2,3-bisphosphoglycerate (2,3-BPG) intermediate.

3. Release of 2-PG:

o The C-3 phosphate of 2,3-BPG is transferred back to the

enzyme’s His, regenerating His-P and releasing 2-PG.

4. Cofactor Role of 2,3-BPG:

o Rarely (~1% of turnovers), 2,3-BPG dissociates, leaving the

enzyme inactive until 2,3-BPG rebinds to rephosphorylate His.

Key Features

 2,3-BPG Dependency:

o Trace 2,3-BPG is essential to maintain enzyme activity

(especially in RBCs, where 2,3-BPG levels are high).

 Energetically Neutral:

o The small ΔG ensures reversibility, allowing the reaction to

adapt to metabolic demands.

Biological Significance

 Prepares for ATP Synthesis: Positions the phosphate on C-2

for enolase (Reaction 9), which creates the high-
energy phosphoenolpyruvate (PEP) for the final ATP-yielding step

Reaction 9 – Enolase Generates a High-Energy Intermediate (PEP)

Key Reaction
  Enzyme: Enolase (requires Mg²⁺).

 Process: Dehydrates 2-phosphoglycerate (2-PG) to

form phosphoenolpyruvate (PEP), a high-energy enol phosphate.

o ΔG°' = +1.8 kJ/mol (slightly unfavorable, but cellular ΔG ≈

0 due to rapid PEP consumption).

o Equilibrium (Keq ≈ 0.5) favors 2-PG, but downstream reactions

pull it forward.

Why PEP is a High-Energy Molecule

 Rearrangement, Not Energy Input:

o 2-PG and PEP have similar total energy, but PEP’s enol-
phosphate group is unstable and releases more energy upon
hydrolysis (ΔG°' = −61.9 kJ/mol for PEP → pyruvate vs. −17.6
kJ/mol for 2-PG → pyruvate).

 Role of Enolase:

o The dehydration traps energy in PEP’s strained enol structure,

making it a potent phosphoryl donor for ATP synthesis in
Reaction 10.

Mechanism & Inhibition

 Dehydration: Removes a water molecule from C-2 and C-3 of 2-PG,

forming the enol double bond.

Biological Significance

 Final ATP Prep: PEP’s high-energy phosphate drives substrate-level

phosphorylation in the next step (pyruvate kinase).

 Regulation Point: Enolase activity is sensitive to cellular energy

needs and inhibited by F⁻ (a glycolytic poison).

Reaction 10 – Pyruvate Kinase Generates the Final ATP of

Glycolysis

Key Reaction
  Enzyme: Pyruvate kinase (PK) (requires Mg²⁺, stimulated by K⁺).

 Process: Transfers phosphate from phosphoenolpyruvate

(PEP) to ADP → pyruvate + ATP.

o ΔG°' = -31.4 kJ/mol (highly favorable; cellular ΔG ≈ -23.0

kJ/mol in erythrocytes).

o Irreversible under physiological conditions (drives

glycolysis forward).

Energetic Payoff

 Net ATP Yield:

o 2 ATP per glucose (since two PEP molecules are produced

per glucose).

o Total glycolysis ATP balance: 2 net ATP (after accounting

for the 2 ATP used in priming).

 Why Such a Large ΔG?

o PEP’s enol group tautomerizes spontaneously to

pyruvate’s more stable keto form, releasing extra energy.

Regulation of Pyruvate Kinase

1. Allosteric Inhibition:

o ATP, acetyl-CoA, alanine (signaling energy surplus or

biosynthetic demand).

2. Allosteric Activation:

o AMP (low energy) and fructose-1,6-bisphosphate

(F1,6BP) (feedforward activation).

3. Covalent Modification (Liver Isozyme):

o Glucagon → cAMP-dependent phosphorylation → inhibits PK,

diverting PEP to gluconeogenesis.

Structural Insight

 Direct Phosphoryl Transfer:

o NMR/EPR studies show PEP’s phosphate is

transferred directly to ADP (no enzyme-bound intermediate).

Biological Significance
  Glycolysis "Payoff": PK ensures the pathway’s net ATP
production (2 ATP/glucose).

 Metabolic Branch Point:

o Pyruvate can enter:

 TCA cycle (aerobic),

 Fermentation (anaerobic, e.g., lactate or ethanol),

 Gluconeogenesis (when PK is inhibited).

What are the products of glycolysis: In addition to 2 ATP, the products of

glycolysis are 2 NADH and 2 pyruvate

What is fermentation: the production of ATP energy by reaction pathways

in which organic molecules function as donors and acceptors of electrons

Presence of oxygen affects and pathways:

 Availability of oxygen determines what happens to pyruvate and

NADH produced in glycolysis
 Absence of oxygen, NADH is recycled to NAD+ by fermentation of
pyruvate
 Presence of oxygen, pyruvate enters the TCA cycle and NADH is
recycled in the electron transport chain

Anaerobic metabolism in yeast and animals:

 in yeast: it is reduced to ethanol

 In other organisms: it is reduced to lactate

Alcoholic fermentation process:

 Pyruvate is decarboxylated to acetaldehyde by pyruvate

decarboxylase in an essentially irreversible reaction because CO2
leaves. Thiamine pyrophosphate is a required cofactor for this
enzyme.
 The second step, the reduction of acetaldehyde to ethanol by NADH,
is catalysed by alcohol dehydrogenase

Lactate fermentation process:

 Lactate dehydrogenase converts pyruvate to lactate and oxidises

NADH
Cons of fermentation: Fermentation leaves much of the chemical energy
in glucose untapped

What happens to lactate: Most lactate is transported via blood to the liver,
where it is converted back to glucose through gluconeogenesis

How do cells regulate glycolysis:

1. Near-Equilibrium Reactions (Reactions 2, 4–9):

o ΔG ≈ 0, meaning these reactions operate close to equilibrium.

o Small changes in metabolite concentrations can easily reverse

or forward these steps.

o Their reversibility allows them to function in both glycolysis

and gluconeogenesis.

2. Irreversible, Regulated Steps (Hexokinase,

Phosphofructokinase, Pyruvate Kinase):

o Large negative ΔG values make these

reactions thermodynamically irreversible in cells.

o These are the key control points of glycolysis, regulated by

allosteric effectors.

o When active, glycolysis proceeds; when inhibited, glycolysis

halts.

3. Gluconeogenesis Bypasses Irreversible Steps:

o Different enzymes are used to reverse reactions 1, 3, and 10

(e.g., glucose-6-phosphatase, fructose-1,6-bisphosphatase,
PEP carboxykinase).

o The near-equilibrium reactions (2, 4–9) remain functional in

both pathways.

Tricarboxylic acid cycle:

1. Entry of Carbon Units:

 o Acetyl-CoA, derived from pyruvate (glycolysis) or fatty acid
oxidation, serves as the entry point for new carbon units into
the cycle.

o Acetyl-CoA (2C) combines with oxaloacetate (4C) via citrate

synthase, forming citrate (6C).

2. Cycle Reactions:

o Citrate is rearranged to isocitrate, which undergoes two

successive decarboxylation, producing α-ketoglutarate
(5C) and then succinyl-CoA (4C).

o Further steps regenerate oxaloacetate, allowing the cycle to

repeat.

3. Carbon Input and Output:

o Carbon enters as acetyl-CoA and exits as CO₂.

o Energy is captured as ATP, NADH, and [FADH₂].

4. Chemical Rationale for the Cycle:

o Direct cleavage of acetate (CH₃COO⁻ → 2 CO₂) is chemically

challenging.

o The TCA cycle cleverly combines condensation with

oxaloacetate and β-cleavage (due to acetate’s lack of a β-
carbon) to facilitate oxidation.

o This process efficiently captures energy while regenerating

intermediates.

What initiates the TCA cycle: citrate synthase

Reaction controlled by citrate synthase:

Acetyl-CoA (2C) + Oxaloacetate (4C) → Citrate (6C) + CoA

Substrate binding to citrate:

  Oxaloacetate binds first, inducing a structural shift that facilitates
acetyl-CoA binding.

 The active site closes to protect the reactive intermediates.

How is citrate synthase regulated:

 Allosteric inhibition by NADH & succinyl-CoA:

o NADH (a product of the TCA cycle) signals high energy status,

slowing the cycle.

o Succinyl-CoA (an intermediate) competes with acetyl-CoA,

providing feedback control.

Why does citrate need to be isomerized to isocitrate by aconitase:

 Problem: Citrate contains a tertiary alcohol, which is difficult to

oxidize without breaking a C–C bond.

 Solution: Aconitase converts citrate into isocitrate (a secondary

alcohol), which is more easily oxidized in subsequent steps.

Oxidative Decarboxylation of Isocitrate to α-Ketoglutarate Reaction

Overview

 Enzyme: Isocitrate dehydrogenase (IDH)

 Substrate: Isocitrate (6C)

 Products:

o α-Ketoglutarate (5C) (via decarboxylation)

o NADH (from NAD⁺ reduction)

o CO₂ (first CO₂ released in the TCA cycle)

 ΔG°': −8.4 kJ/mol (drives the cycle forward).

Two step mechanism of the reaction from isocitrate to α-Ketoglutarate:

1. Oxidation:
 o The C-2 alcohol of isocitrate is oxidized to a ketone,
forming oxalosuccinate (an unstable β-keto acid).

2. Decarboxylation:

o Oxalosuccinate undergoes β-decarboxylation, losing a

carboxyl group as CO₂ and yielding α-ketoglutarate.

How is isocitrate dehydrogenase regulated:

 Allosteric Inhibition:

o NADH (signaling high energy)

o ATP (energy surplus)

 Allosteric Activation:

o ADP (signaling low energy, increases enzyme affinity for

isocitrate by 10x)

Oxidative decarboxylation of ketoglutarate to succinyl-CoA

 Enzyme: α-Ketoglutarate dehydrogenase complex (KDHC)

 Substrate: α-Ketoglutarate (5C) + CoA-SH + NAD⁺

 Products:

o Succinyl-CoA (4C) (high-energy thioester)

o NADH (from NAD⁺ reduction)

o CO₂ (second CO₂ released in the TCA cycle)

 ΔG°': ~ -33.5 kJ/mol (highly exergonic, irreversible).

Biological significance of Oxidative decarboxylation of ketoglutarate to

succinyl-CoA: Critical Energy Yield: Produces NADH (→ ETC) and succinyl-
CoA (→ substrate-level phosphorylation).

Significance of Succinyl-CoA Synthetase: Substrate-Level Phosphorylation:

Reaction of Succinyl-CoA Synthetase:

- Enzyme: Succinyl-CoA synthetase (also called succinate thiokinase).

- Substrate: Succinyl-CoA + GDP (or ADP in plants/bacteria) + Pᵢ.
- Products: Succinate + GTP (or ATP) + CoA.
- ΔG°': −3.3 kJ/mol (driven by thioester hydrolysis; reversible but
favors GTP/ATP formation).

Summary of the First Half of the TCA Cycle (Acetyl-CoA → Succinate)

 Input: 1 acetyl-CoA (2C).

 Output (per acetyl-CoA):

o 2 CO₂ (from isocitrate & α-ketoglutarate dehydrogenase).

o 2 NADH (from isocitrate & α-ketoglutarate dehydrogenase).

o 1 GTP/ATP (from succinyl-CoA synthetase).

o 1 succinate (4C).

Completing the Cycle: Succinate → Oxaloacetate

The remaining steps regenerate oxaloacetate through:

1. Oxidation: Succinate → Fumarate (via succinate dehydrogenase,

reduces [FAD] to [FADH₂]).

2. Hydration: Fumarate → Malate (via fumarase).

3. Oxidation: Malate → Oxaloacetate (via malate dehydrogenase,

reduces NAD⁺ to NADH).

Products of the second half of the TCA cycle

 Electron Carriers:

o 1 [FADH₂] (from succinate dehydrogenase, enters ETC at

Complex II).

o 1 NADH (from malate dehydrogenase).

 Analogous to Fatty Acid β-Oxidation: Similar

oxidation/hydration/oxidation steps occur in fatty acid breakdown.
Key TCA Cycle Intermediates & Their Biosynthetic Roles

1. α-Ketoglutarate

o Converted to glutamate via transamination.

o Precursor for:

 Proline, arginine, glutamine (amino acids).

 Purines (DNA/RNA synthesis).

2. Succinyl-CoA

o Major carbon source for heme (porphyrin) synthesis (e.g.,

hemoglobin, cytochromes).

3. Oxaloacetate (OAA)

o Converted to aspartate (transamination).

o Aspartate is a precursor for:

 Pyrimidines (nucleotide synthesis).

 Asparagine, methionine, lysine, threonine,

isoleucine.

o Can be decarboxylated to PEP (phosphoenolpyruvate),

which fuels:

 Aromatic amino acids (phenylalanine, tyrosine,

tryptophan).

 Serine, glycine, cysteine (via 3-phosphoglycerate).

 Gluconeogenesis (glucose synthesis).

4. Citrate

o Exported from mitochondria and cleaved by ATP-citrate

lyase into:

 Acetyl-CoA (for fatty acid synthesis).

 Oxaloacetate, which can be recycled as:

 Malate → re-enters mitochondria.

 Pyruvate (via malic enzyme) → reused for energy

or biosynthesis.
What is the purpose of anaplerotic reactions: The TCA cycle loses
intermediates when they are siphoned off for biosynthesis. To maintain
cycle function, cells rely on anaplerotic reactions ("filling up" reactions),
which replenish these intermediates.

Key Anaplerotic Reactions

1. Pyruvate Carboxylase (Most Important in Animals)

o Reaction: Pyruvate+CO2+ATP→Oxaloacetate (OAA)+ADP+Pi

o Location: Mitochondria (animals only).

o Regulation:

 Allosterically activated by acetyl-CoA (ensures TCA cycle

continues when acetyl-CoA is abundant).

2. PEP Carboxylase (Plants, Bacteria, Yeast)

 Reaction:

PEP+CO2→Oxaloacetate+PiPEP+CO2→Oxaloacetate+Pi

 Regulation:

o Inhibited by aspartate (feedback control to prevent excess

OAA → aspartate).

Malic Enzyme (Animals & Plants)

 Reaction:

Pyruvate+CO2+NADPH→Malate+NADP+Pyruvate+CO2
+NADPH→Malate+NADP+

 Role:

o Provides malate, which can enter mitochondria and convert

back to OAA.

3. Malic Enzyme (Animals & Plants)

o Reaction:
Pyruvate+CO2+NADPH→Malate+NADP+Pyruvate+CO2
+NADPH→Malate+NADP+

o Role:

 Provides malate, which can enter mitochondria and

convert back to OAA.

 Also generates NADPH (useful for biosynthetic pathways

like fatty acid synthesis).

4. Amino Acid Catabolism

o Breakdown of proteins provides TCA cycle intermediates:

 Pyruvate (from alanine, serine, cysteine).

 Acetyl-CoA (from leucine, lysine).

 α-Ketoglutarate (from glutamate, glutamine, arginine,

proline).

 Succinyl-CoA (from isoleucine, valine, methionine).

 Fumarate (from phenylalanine, tyrosine).

 Oxaloacetate (from aspartate, asparagine).

Regulation of TCA cycle;

Activator Regulatory
Enzyme Reaction Inhibitors
s Logic

Prevents
Acetyl- excess entry of
Citrate CoA + - NADH (high - ADP (low acetyl-CoA
Synthase OAA → energy) energy) when energy
Citrate (NADH/ATP) is
high.

- Succinyl-
CoA
(feedback)

- ATP
 Activator Regulatory
Enzyme Reaction Inhibitors
s Logic

(energy
surplus)

Isocitrate - NADH - ADP, Links cycle rate

Isocitrate
→ α-KG + (excess NAD⁺ to cellular
Dehydrogena
CO₂ + reducing (energy ATP/NAD⁺
se
NADH equivalents) demand) needs.

- ATP - Ca²⁺ Ca²⁺ couples

(energy (muscle metabolism to
surplus) activity) contraction.

α-KG →
α- - NADH, Ensures
Succinyl- - Ca²⁺
Ketoglutarate Succinyl-CoA balanced flux
CoA + (parallel
Dehydrogena (product through late-
CO₂ + activation)
se feedback) cycle steps.
NADH

Regulation of pyruvate dehydrogenase (PDH) importance:

- pyruvate dehydrogenase (PDH) is a critical metabolic gatekeeper

that converts pyruvate → acetyl-CoA, committing carbon to either,
- Since animals cannot convert acetyl-CoA back to glucose, PDH
regulation ensures carbon is directed appropriately based on cellular
needs.

Purpose of glyoxylate cycle:

 Converts 2 acetyl-CoA → 1 oxaloacetate (OAA) without losing carbon

as CO₂.

 Enables gluconeogenesis from fats (critical for seedlings growing in

darkness).

Key Differences from the TCA Cycle:

Feature TCA Cycle Glyoxylate Cycle

2 CO₂ per cycle

CO₂ Release No CO₂ lost
(decarboxylation)

Acetyl-CoA
1 per cycle 2 per cycle
Use

Net Output Energy (ATP, NADH) OAA for biosynthesis

5 reactions (bypasses
Steps 8 reactions
decarboxylation)

Unique Enzymes:

- Isocitrate Lyase: Splits isocitrate → succinate + glyoxylate (instead

of α-KG + CO₂).

- Malate Synthase: Combines glyoxylate + acetyl-CoA

→ malate (replenishes 4C intermediates).

1. Organelle Localization:

o Plants: Glyoxysomes (specialized peroxisomes).

o Bacteria/Algae: Cytoplasm.

o Mitochondrial Collaboration: Glyoxysomes lack 3 TCA enzymes

(succinate DH, fumarase, malate DH), so they shuttle
intermediates to/from mitochondria.

Regulation

 Inhibited by Glucose: Avoids unnecessary acetate use when sugars

are available.

 Activated by Acetyl-CoA: Signals fatty acid breakdown (e.g., during

starvation).
Why Animals Lack This Cycle

Animals cannot synthesize glucose from acetyl-CoA because they:

1. Lack isocitrate lyase and malate synthase.

2. Prioritize energy production (TCA cycle) over carbon conservation.

Where does oxidative phosphorylation take place : in the mitochondria

Mitochondrial structure:

1. Outer Membrane

o Contains porin proteins, allowing free diffusion of small

molecules

2. Intermembrane Space

o Contains enzymes like creatine kinase and adenylate kinase.

3. Inner Membrane

o Highly folded into cristae, increasing surface area.

o Houses electron transport chain (ETC) proteins and ATP

synthase.

4. Matrix

o Contains enzymes for the TCA cycle (except succinate

dehydrogenase, which is in the inner membrane) and fatty
acid oxidation.

o Has its own circular DNA, ribosomes, and protein-synthesis

machinery, though most mitochondrial proteins are nuclear-
encoded.

Reduction Potentials and Free Energy Changes in Redox Reactions:

 NADH and FADH₂ are high-energy electron carriers that tend to be

oxidized (lose electrons).
  Oxidative phosphorylation converts the energy from electron
transfer into ATP (stored as phosphoanhydride bonds).

with lower ℰ°′ (e.g., NADH) to those with higher ℰ°′ (e.g., O₂),
 In the electron transport chain (ETC), electrons move from carriers

releasing energy used to pump protons and synthesize ATP.

Key Functions of the ETC

 Reoxidizes NADH and FADH₂ (from glycolysis, TCA cycle, fatty acid
oxidation).

 Multiple protein complexes (I-IV) and mobile carriers (CoQ,

cytochrome c) shuttle electrons

 Transfers electrons to oxygen (O₂), the final electron acceptor,

forming water (H₂O).

 Uses the energy released to pump protons (H⁺), creating a proton

gradient for ATP synthesis

The ETC can be isolated into four protein complexes:

1. Complex I (NADH-CoQ Reductase)

o Accepts electrons from NADH (linked to glycolysis, TCA cycle,

fatty acid oxidation).

o Transfers electrons to ubiquinone (CoQ), producing UQH₂.

2. Complex II (Succinate-CoQ Reductase)

o Contains succinate dehydrogenase (from TCA cycle).

o Directly reduces CoQ to UQH₂ (bypasses Complex I).

3. Complex III (CoQ-Cytochrome c Reductase)

o Oxidizes UQH₂ and transfers electrons to cytochrome c.

o Uses the Q cycle to maximize proton pumping.

4. Complex IV (Cytochrome c Oxidase)

o Accepts electrons from cytochrome c.

o Reduces O₂ to H₂O (terminal step).

 o Contains cytochrome a/a₃ and copper centers.

Where does proton pumping occur:

 Proton pumping occurs at Complexes I, III, and IV, generating the H⁺

gradient for ATP synthesis.

 Complex II does not pump protons but feeds electrons into the
system.

How the Proton Gradient Forms

1. Electron Transport Chain (ETC) Pumps Protons

o As electrons move through Complexes I, III, and IV, protons

(H⁺) are pumped from the matrix → intermembrane space.

o Creates:

 pH gradient (matrix more alkaline, intermembrane

space more acidic).

 Electrical gradient (matrix negative, intermembrane

space positive).

2. Electrochemical Potential

o The combined pH + charge gradient = proton-motive force

(PMF).

o Energy stored in PMF drives ATP synthase (H⁺ flow back into
matrix powers ATP production).

Thermodynamic Implications of Chemiosmotic Coupling:

 Outward proton pumping (matrix → intermembrane space) is

energetically unfavorable (ΔG = +23.3 kJ/mol), requiring energy
from electron transport.

 Inward proton flow (intermembrane space → matrix) releases 23.3

kJ/mol, which drives ATP synthesis via ATP synthase.

 ATP synthase couples proton flow (ΔG = -23.3 kJ/mol)

to phosphorylation (ΔG°’ for ATP synthesis ≈ +30.5 kJ/mol).
 Under physiological conditions, ~3–4 protons are needed per ATP
synthesized.
Structure of ATP Synthase:

ATP synthase is a rotary molecular motor with two main parts:

1. F₁ Unit (Catalytic Head)

 Location: Matrix-facing "knobs" seen in electron microscopy.

 Subunits: α₃β₃γδε (hexameric ring).

o Three catalytic β-subunits (bind ADP + Pᵢ → ATP).

o Three non-catalytic α-subunits (structural).

 Function: Synthesizes ATP via binding change mechanism.

2. F₀ Unit (Proton Channel)

 Location: Embedded in the inner mitochondrial membrane.

 Subunits: a₁b₂c₁₀₋₁₅ (forms a rotor-stator system).

o c-ring: 10–15 subunits; rotates as protons flow through.

o a-subunit: Guides protons into/out of the c-ring.

o Stator (b, d, OSCP): Anchors F₁ to F₀, preventing rotation.

Mechanism of ATP Synthesis - The Binding Change Model:

1. Proton Flow Drives Rotation

o Protons move down their gradient through F₀, causing the c-

ring to rotate.

o Each proton binds to a c-subunit, driving a 120° turn per 3–4

protons.

2. Conformational Changes in F₁

o Rotation of the γ-subunit (central stalk) induces three states in

the β-subunits:

 Open (O): Releases ATP.

 Loose (L): Binds ADP + Pᵢ.

 Tight (T): Squeezes ADP + Pᵢ into ATP.

 3. ATP Release

o Each full rotation (360°) produces 3 ATP molecules

Affinity of F1 3 conformations:

 Open – low affinity for ligands; inactive

 Loose – increased affinity for ligands – trapped in the subunit;
inactive
 Tight – high affinity for ligand; active

Disruptors of ETC and ATP synthesis:

 Inhibitors of the ETC reduce proton gradient formation

 Inhibitors of ATP synthase prevents ATP synthesis
 Uncouplers dissipate the gradient without forming ATP (energy lost
as heat)
 Thermogenin is an endogenous uncoupler

Purpose of glycerophosphate shuttle: allows cytosolic NADH (from

glycolysis) to contribute to mitochondrial ATP production, even though
NADH itself cannot cross the mitochondrial membrane.

1. Cytosolic Step (NADH Oxidation)

o Enzyme: Glycerol-3-phosphate dehydrogenase

(cytosolic, NAD⁺-dependent).

o Reaction:

Dihydroxyacetone phosphate (DHAP)+NADH+H+→Glycerol-3-phosphate (

G3P)+NAD+

o Converts NADH → NAD⁺, regenerating glycolysis cofactors.

2. Mitochondrial Step (FAD Reduction)

o Enzyme: Glycerol-3-phosphate dehydrogenase

(mitochondrial, FAD-dependent, membrane-bound).

o Reaction:

G3P+FAD→DHAP+FADH2
 o FADH₂ transfers electrons directly to ubiquinone (UQ),
forming UQH₂, which enters the ETC at Complex II.

Energy Yield

 1.5 ATP per NADH (since FADH₂ bypasses Complex I, reducing

proton pumping).

 Trade-off: Lower ATP yield (vs. 2.5 ATP via malate-aspartate

shuttle) but faster and irreversible, ensuring NADH oxidation
even at low [NADH]/[NAD⁺] ratios.

Purpose of malate-aspartate shuttle: efficiently transfers electrons

from cytosolic NADH into the mitochondrial matrix,
generating NADH (instead of FADH₂) for the ETC, maximizing ATP yield
(2.5 ATP/NADH).

Process of malata-aspartate shuttle:

1. Cytosolic Steps

1. Reduction of Oxaloacetate (OAA)

o Enzyme: Cytosolic malate dehydrogenase (MDH).

o Reaction:

OAA+NADH+H+→Malate+NAD+OAA+NADH+H+→Malate+NAD+

o Converts NADH → NAD⁺, allowing glycolysis to continue.

2. Malate Transport

o Malate crosses the inner mitochondrial membrane via

the malate-α-ketoglutarate antiporter.

2. Mitochondrial Steps

3. Oxidation of Malate

o Enzyme: Mitochondrial malate dehydrogenase (MDH).

o Reaction:

Malate+NAD+→OAA+NADHMalate+NAD+→OAA+NADH

o Regenerates NADH in the matrix, which feeds into Complex

I (higher ATP yield).
Benefits of malate aspartate shuttle compared to glucophosphate shuttle:

1. Reversibility

o Unlike the glycerophosphate shuttle, this system can run

backward if mitochondrial [NADH] is higher than cytosolic
[NADH].

2. Higher Energy Yield

o Produces mitochondrial NADH → ~2.5 ATP per NADH (vs.

1.5 ATP via glycerophosphate shuttle).

Cell signalling:

Type of hormone chemical species:

 Steroid hormones
 Amino Acid derivatives
 Peptide hormones

Hormone and receptor affinities: Hormones and other signal molecules in

biological systems bind with very high affinities to their receptors,
displaying K values in the range of 10-12 to 10-6 M.

Two ways in which steroid hormones act:

 First, by entering cells and migrating to the nucleus, steroid

hormones act as transcription regulators, modulating gene
expression. (slow)
 Steroids can also act at the cell membrane, (rapid)

What is signal transduction:

consists of a stepwise progression of signalling stages: receptor⎯→

transducers⎯→effectors. The receptor perceives the signal, transducers
relay the signal, and the effectors convert the signal into an intracellular
response.
Significance of enzyme cascades: Enzyme cascades function as biological
amplifiers, ensuring swift, specific, and large-scale cellular responses.

Glucocorticoids and the glucocorticoids receptor (GR):

Glucocorticoids are steroids, One key receptor for glucocorticoids is the
glucocorticoid receptor (GR), which functions as a ligand-activated
transcription factor.

GR protein structure:

 Activation Domain (AD): It helps facilitate the initiation of

transcription once the receptor is bound to DNA.
 DNA Binding Domain (DBD): This domain contains zinc finger
motifs, which are specialized structural motifs that stabilize the
protein’s interaction with DNA.
 Ligand-Binding Domain: This region binds the steroid hormone (the
ligand)

Regulation of GR protein:

 In the absence of hormone, the GR remains in the cytoplasm, where

it is sequestered by a complex of inhibitory proteins. These
chaperones maintain the receptor in an inactive state and prevent it
from translocating to the nucleus.

 When a glucocorticoid hormone enters the cell and binds to the LBD
of GR, the receptor undergoes a conformational shift that causes it
to dissociate from the inhibitory proteins. This activation enables the
GR to translocate into the nucleus.

Three receptor superfamilies:

G-protein–coupled receptors

single-transmembrane segment (1-TMS) catalytic receptors

Oligomeric ion channels

The G-protein–coupled receptors: are integral membrane proteins with an
extracellular recognition site for ligands and an intracellular recognition
site for a GTP-binding protein

The single-transmembrane segment (1-TMS) catalytic receptors are

proteins with only a single transmembrane segment and substantial
globular domains on both the extracellular and the intracellular faces of
the membrane

What are the domains in 1-TMS: The extracellular domain is the ligand
recognition site and the intracellular catalytic domain is either a tyrosine
kinase or a guanylyl cyclase

Oligomeric ion channels consist of associations of protein subunits, each

of which contains several transmembrane segments. These oligomeric
structures are ligand-gated ion channels.

What are the ligands for the ion channels: neurotransmitters

GPCR Signal Transduction via G Proteins

1. G Protein Structure and Activation

 Heterotrimeric G proteins consist of:

o Gα (binds GDP/GTP, has GTPase activity)

o Gβγ (dimer, stabilizes inactive state)

 Mechanism of Activation:

o Inactive state: Gα binds GDP, complexed with Gβγ.

o Hormone binding to GPCR →GDP replaced by GTP on Gα.

o Gα(GTP) dissociates from Gβγ → binds effector proteins (e.g.,

adenylyl cyclase).

o Termination: Gα hydrolyses GTP → GDP → reassembles with

Gβγ (inactive state).

2. Downstream Signalling & Amplification

 Effector Activation:

o Gα (stimulatory): Activates adenylyl cyclase → cAMP

production.
 o Gαi (inhibitory): Suppresses adenylyl cyclase → reduces cAMP.

 Signal Amplification:

o 1st stage: Single hormone-bound GPCR activates multiple G

proteins.

o 2nd stage: Each Gα(GTP)-activated adenylyl cyclase

produces many cAMP molecules.

1. Downstream Effects (via G Proteins):

o Activation of enzymes:

 Adenylyl cyclase → produces cAMP (from ATP).

 Guanylyl cyclase → produces cGMP (from GTP).

 Phospholipases → generate lipid-derived second

messengers.

Muscarinic acetylcholine receptor (Activation of receptor-coupled G

proteins):

 In heart muscle, the muscarinic acetylcholine receptor activates its

effector K+ channel via the Gβγ subunit of a Gi protein.
 Binding of acetylcholine triggers activation of the G αi subunit and its
dissociation from the Gβγ subunit in the usual way.
 In this case, however, the released Gβγ subunit (rather than Gαi·GTP)
binds to and opens the associated effector protein, a K + channel.
 The increase in K+ permeability hyperpolarizes the membrane,
which reduces the frequency of heart muscle contraction.
 Activation is terminated when the GTP bound to G αi is hydrolyzed (by
a GAP enzyme that is an intrinsic part of the G αi subunit) to GDP and
Gαi·GDP recombines with Gβγ

Single TMS receptor characteristics:

1. Structure:

o Single transmembrane segment (1-TMS) receptors with:

 Extracellular ligand-binding domain (recognizes signals).

 Intracellular enzymatic domain (mediates signalling).

 2. Two Major Types:

o Receptor Tyrosine Kinases (RTKs):

o Receptor Guanylyl Cyclases (RGCs):

Receptor Tyrosine kinases:

Structure:

 Extracellular Domain: Responsible for ligand binding

 Single Transmembrane Segment: A single alpha-helical hydrophobic
domain that anchors the receptor in the plasma membrane.
 Intracellular (Cytoplasmic) Domain: tyrosine kinase domain, which is
responsible for the enzymatic activity—it transfers phosphate
groups from ATP to tyrosine residues on proteins.

Mechanism of Activation

1. Ligand Binding:

o A specific ligand (such as a growth factor) binds to the

extracellular domain of the RTK.

2. Receptor Dimerization:

o Ligand binding induces dimerization (pairing) of two RTK

molecules.

o This brings the intracellular kinase domains into close

proximity.

3. Autophosphorylation:

o The kinase domains phosphorylate tyrosine residues on each

other (cross-phosphorylation).

o This increases the receptor’s kinase activity and creates

phosphotyrosine docking sites.

4. Intracellular proteins bind to these phosphotyrosine sites, this

initiates downstream signaling pathways

Example: Epidermal Growth Factor Receptor (EGFR)

Receptor guanylyl cyclases:

Structure

 Extracellular Domain: Binds to a specific ligand This binding initiate

receptor activation.
  Single Transmembrane Segment (TMS): Anchors the receptor in the
plasma membrane.
 Intracellular Domain: Guanylyl Cyclase Catalytic Domain: Catalyses
the conversion of GTP to cGMP upon activation.

Mechanism of Activation

1. Ligand Binding:

o A peptide hormone (e.g., ANP) binds to the extracellular

domain of the receptor GC.

2. Conformational Change:

o Ligand binding induces a structural change that is transmitted

through the single TMS to the intracellular domain.

3. Activation of Catalytic Domain:

o The conformational signal activates the guanylyl cyclase

domain, leading to production of cGMP from GTP

Example:

Natriuretic Peptide Receptors (RGCs):

o ANP/BNP (heart-derived) reduce blood pressure by promoting

kidney salt/water excretion.

What happens when receptors are activated in Oligomeric ion channels:

Binding of hormone causes the pore to open

3 ways in which receptor signals are transduced:

 Exchange of GDP for GTP by GTP-binding proteins

 Receptor-mediated activation of phosphorylation cascades that in
turn trigger activation of various enzymes
 Conformation changes that open ion channels or recruit proteins
into nuclear transcription complexes

What are the three most common second messengers:

 Cyclic nucleotides e.g. cyclic AMP

  Phospholipid breakdown products

 Calcium

Cyclic AMP (cAMP) as a Second Messenger

Regulation of cAMP Production

 Adenylyl Cyclase (AC):

o Produces cAMP

o Activated by Gαs-GTP (stimulatory G protein subunit), which

binds at a site distant from the catalytic centre.

 Termination:

o Gα’s intrinsic GTPase activity → hydrolysis to GDP →

inactivation  G dissociates with Adenyl cyclase  G
reassociates with receptor

o Phosphodiesterase converts cAMP to AMP

Downstream Effects of adenylate cyclase activation Protein Kinase A (PKA)

 cAMP’s primary target: Protein Kinase A (PKA).

o Inactive when regulatory subunits are bound

o Binding of cAMP to PKA’s regulatory subunits releases catalytic

subunits → phosphorylation of downstream proteins.

 Amplification: A single hormone signal → massive cAMP

production → widespread PKA activation.

Protein phosphorylation:

 Kinases add phosphate groups to specific amino acids –

serine/threonine, tyrosine
 Phosphatases remove phosphate groups

Phospholipase-Mediated Second Messenger Production

1. Phospholipid-Derived Second Messengers

  Membrane phospholipids (e.g., phosphatidylinositol, PIP₂) are
cleaved by phospholipases to generate key second messengers:

o Phospholipase C (PLC) hydrolyzes PIP₂ → produces:

 IP₃ (inositol trisphosphate): Water-soluble, triggers Ca²⁺

release from intracellular stores.

 DAG (diacylglycerol): Membrane-bound,

activates protein kinase C (PKC).

2. Regulation of Phospholipase C (PLC) Isozymes

 PLC-β: Activated by G proteins (e.g., GPCR signaling via Gαq).

 PLC-γ: Activated by receptor tyrosine kinases (RTKs) (e.g., growth

factor receptors).

 Common Features:

o All PLC isoforms require Ca²⁺ for activity.

3. Downstream Signalling Effects

 IP₃ Pathway:

o Binds ER IP₃ receptors → releases Ca²⁺ → regulates enzymes

Calcium as a second messenger

1. Role of Calcium (Ca²⁺) in Signalling: binds to Protein kinase C and

Ca+2 protein kinase, which activates target proteins

2. Sources of Cytoplasmic Ca²⁺ Increase

o Extracellular influx:

 cAMP can open plasma membrane Ca²⁺ channels,

allowing external Ca²⁺ entry.

o Intracellular release:

 IP₃ (from PIP₂ hydrolysis) binds to ER/IP₃ receptors →

releases Ca²⁺ from stores.

Rhodopsin GPCR involved in vision:

Rhodopsin is a GPCR

 Protein component – opsin

 G protein – transducin
 Pigment – retinal (absorbs light)

How Rhodopsin Works in Light and Dark

In the Dark (No Light):

1. Rhodopsin is inactive (with 11-cis-retinal bound).

2. cGMP levels are high → keeps Na⁺/Ca²⁺ channels open. Rapid

neurotransmitter releases

3. Rod cells are depolarized (positively charged).

In Bright Light:

1. Light hits retinal → converts 11-cis → all-trans-retinal.

2. Rhodopsin changes shape and activates transducin (G-protein).

3. Transducin activates phosphodiesterase → breaks down cGMP.

4. Na⁺/Ca²⁺ channels close → rod cell hyperpolarizes (negatively

charged). Slow neurotransmitter release

Reset (Back to Dark):

 Retinal detaches, opsin resets.

 cGMP is restored → channels reopen.

 Rod cell depolarizes → ready for the next light signal

What are the Mitogen-Activated Protein kinases (MAPK):

• Three-tiered kinase signaling pathways that function in

phosphorelay systems:

MAPK kinase phosphorylates MAPK kinase

MAPK kinase phosphorylates MAPK

 MAPK phosphorylates range of targets including other kinases &
transcription factors

Phosphorelay system basic pathway:

Stimulus  activator  MKKK  MKK  MAPK  Substrate

Ras Phosphorelay system basic pathway:

Growth factor  RasGTP  c-RAF 1  MKK 1  ERK 1  p90RSK

What is Ras and what makes it unique:

Ras is a monomeric G-protein (also called a small GTPase) that functions

as a molecular switch in the MAPK pathway. Unlike heterotrimeric G-
proteins activated by GPCRs, Ras is activated downstream of Receptor
Tyrosine Kinases (RTKs).

Ras activation process:

 Ligand binds to an RTK, causing receptor dimerization and

autophosphorylation.
 Adaptor proteins bind to phosphotyrosine residues on the RTK.
 Adaptor proteins recruit’s guanine nucleotide exchange factor (GEF).
 GEF binds to Ras, which Induces conformation change which favours
GTP binding
 Ras undergoes a conformational change, switching to its active,
GTP-bound form.
 GTPase activating protein (GAP) Induces conformational change in
Ras to increase the rate of GTP hydrolysis within Ras
 Active Ras recruits and activates Raf (MAPKKK), initiating the MAPK
cascade.
 Ras is the deactivated

What does Ras being a proto-oncogene mean: Proto-oncogenes are

normal genes that regulate cell growth and division

What are oncogenes: proto-oncogenes that are mutated, they can become
oncogenes, contributing to uncontrolled proliferation and cancer.
Oncogenic ras pathway:

 Point mutations in ras can impair Ras's GTPase activity.

 This causes Ras to remain in the GTP-bound "on" state, permanently
activating downstream pathways, including MAPK.
 This leads to uncontrolled cell growth, resistance to apoptosis, and
ultimately tumor formation.’

Nicotinic Acetylcholine Receptor: directly mediates ion flow across

membranes in response to neurotransmitter binding.

Electrical Signaling in Neurons:

 Neuronal communication is primarily electrical, based on transient

changes in membrane potential.

 This electrical activity is generated and propagated by ion gradients

across the plasma membrane.

Process of Signal:

Action Potential and Neurotransmitter Release

 A stimulus causes voltage-gated Na⁺ channels to open, allowing Na⁺

influx, depolarizing the membrane.
 This influx causes opening of voltage-gated Ca²⁺ channels.
 Ca²⁺ enters the cell, triggering synaptic vesicles containing
acetylcholine (ACh) to fuse with the presynaptic membrane.
 ACh is released into the synaptic cleft via exocytosis.

Activation of the Nicotinic ACh Receptor

 ACh diffuses across the synaptic cleft and binds to nAChR on the
postsynaptic membrane. ACh diffuses across the synaptic cleft and
binds to nAChR on the postsynaptic membrane
 Upon activation, the receptor's central pore opens, allowing ions to
pass through.
 Na⁺ flows into the cell (down its steep concentration gradient), and
K⁺ flows out. Because the Na⁺ influx is greater, the membrane
depolarizes, generating an excitatory postsynaptic potential
Termination of Signal

 The channel remains open only for a few milliseconds before it

closes, even if ACh is still present.
 The enzyme acetylcholinesterase (AChE), present in the synaptic
cleft, rapidly breaks down ACh into acetate and choline.
 As the concentration of ACh drops, it dissociates from the receptor,
which returns to its resting (closed) state. The receptor is now ready
to respond to the next wave of neurotransmitter.

Recombination:

What is Recombinant DNA molecules: constructions of molecules using

sequences from different sources.

How is a recombinant DNA molecule produced:

A fragment of DNA that includes the gene to be cloned is inserted into a

circular DNA molecule called a vector to produce a recombinant DNA
molecule

What is a vector: DNA molecule, often a plasmid or virus, used to carry

and deliver a specific DNA segment into a host cell

Vector replication within host:

• Within the host cell, the vector multiplies, producing numerous

identical copies of itself and the gene that it carries.

• When the host cell divides, copies of the recombinant DNA molecule
are passed to the progeny, and further vector replication occurs.

When is the recombinant DNA molecule considered to be cloned: After cell

division, a colony, or clone, of identical host cells is produced; each cell in
the clone contains one or more copies of the recombinant DNA molecule

Basic steps of recombinant DNA technology:

• Isolate the DNA (copy, PCR)

• Cut DNA at Known palindromic cleavage sites using RE

• Use Ligase (paste) to glue back together into new combinations

 • Introduce the recombined (hence “recombinant”) DNA into an
organism for replication and reproduction

• Select for the desired genotype/phenotype

Two types of DNA:

Total cell DNA: contain all genomic DNA and extracellular DNA

Pure vector DNA: comes in the form of a plasmid or phage, where the
plasmid DNA is separated from the total DNA

Extraction of total DNA:

1.) Cell lysis: weakening of cell wall with EDTA or SDS, allows for the
exposure of cell extract, where DNA can be centrifuged out
2.) Removal of protein contaminants within cell extract: mix extract
with phenol, centrifuge sample, layers of DNA – protein – phenol
appear
3.) Concentration of DNA: ethanol precipitates polymeric nucleic acid,
which can be collected by centrifuging

Plasmid purification :

 Alkaline lysis method: there is a narrow pH range at which non-

supercoiled DNA is denatured, whereas supercoiled plasmids are
not. pH is adjusted to 12.0–12.5 by NaOH, which allows for the
denaturing of genomic DNA
 If acid is now added, these denatured bacterial DNA strands
reaggregate into a tangled mass. The insoluble network can be
pelleted by centrifugation (and insoluble RNA and proteins),
leaving plasmid DNA in the supernatant

What is the basic regulatory unit of information transfer: restriction

enzymes

What are restriction enzymes: endonucleases, proteins that act like

molecular scissors, cutting DNA at specific, often palindromic, sequences

Why do endonucleases/RE cut at palindromic sequences :

Restriction enzymes are homodimers, made up of two identical protein

subunits. This means that they both recognise the same sequence.
Because the recognition sequence is the same on both strands in
palindromic sequences, the enzyme can cut both strands of the DNA at
the same

Type 2 restriction enzymes: requires no ATP, hydrolytic cleavage of

particular phosphodiester bonds in the DNA sequence 4 – 8 bp long and
palindromic

Most commonly used Type 2 RE : Type IIP

Characteristics of Type IIP restriction enzymes:

 Recognise palindromic sequence

 Each has a known sequence of 4 to 8 base pairs.
 Can recognise and cut DNA at recognition sites.
 Make a blunt or a sticky end.
 Require Mg+2 as a co factor

What are restriction fragments: The pieces of DNA produced

Sticky ends vs blunt ends:

Sticky ends: Occurs when RE cuts at opposing positions between the

complementary strands, generating short overhangs

Blunt ends: a simple double-stranded cut in the middle of the recognition

sequence
Why are sticky ends called sticky: Called sticky/cohesive overhangs as
base pairing of single stranded DNA tails are highly likely to bind
together again (“becomes sticky”).

Using One Restriction Enzyme (Single Cut Cloning)

 Issue: The vector and insert are both cut with only one enzyme,
creating identical ends.

 Consequence:

o Non-directional cloning: The insert can ligate in either

orientation.

o Vector self-ligation: Since the ends are compatible (identical),

the vector can re-ligate without an insert, leading to high
background of parental (empty) plasmid colonies.

Using Two Compatible Enzymes (Double Digestion)

 Compatible Cohesive Ends are produced when you have two

separate enzymes that recognize very similar sequences.

 Issue: Despite being from different enzymes, the ends can ligate
together.

 Consequence:

o Still non-directional, if both ends of the vector and insert are

compatible and indistinguishable after ligation.

o Religation can still occur, leading to background colonies.

Cut the vector and insert with two different enzymes that cannot re-ligate:

 Advantages:

o A vector can’t re-circularise

o Will get low background - only a few parental vectors

o Target DNA is inserted in one orientation

o Can use compatible ends

DNA Ligase Function

DNA ligase catalyzes the formation of phosphodiester bonds between the

5’ phosphate of one DNA or RNA strand and the 3’ hydroxyl of another,
covalently joining DNA fragments.

 Forms two covalent bonds to link the donor (5' phosphate) and
acceptor (3' hydroxyl) ends.

 ATP is required, and the reaction occurs in three steps:

1. Adenylation of the enzyme (AMP added, pyrophosphate

released).

2. Transfer of AMP to the 5’ phosphate of the donor.

3. Phosphodiester bond formation between the donor and

acceptor strands.

When DNA is cut with the same restriction enzyme, matching sticky ends
allow for efficient ligation.

What is an expression vector: a type of plasmid (circular DNA molecule)

designed to introduce and express a gene of interest (GOI) in a target host
cell.

4 Key Elements of a Typical Expression Vector

1. Gene of Interest (GOI) Expression Cassette

o Includes a promoter, GOI, and a transcription terminator or

poly(A) signal for proper expression and mRNA stability.

2. Host Origin of Replication (ori)

o Allows the plasmid to replicate in bacteria.

o Determines host range and copy number in E. coli or other

species.

3. Selection Marker for Host Cells

o Confers resistance to antibiotics like blasticidin S or

hygromycin B.

o Ensures only host cells with the plasmid survive.

Blue-White Screening Mechanism:

Component Function/Effect

Host with defective β-galactosidase (lacking α-

lacZΔM15 E. coli
peptide)

pUC vector Provides the α-peptide for complementation

Functional lacZ Only formed when vector is intact (no insert)

Insertional GOI inserted into MCS disrupts lacZα → no

Inactivation complementation

IPTG Induces expression of lacZ gene (allolactose analog)

Colorless substrate cleaved by β-galactosidase →

X-gal
blue product

🧬 Outcome of Transformation:

LacZα β-Galactosidase Colony

Vector Type
Functional? Activity Color

Empty vector Yes Active Blue

Recombinant vector
No Inactive White
(insert)
Polymerase chain reaction requirements:

 template DNA: Double-stranded DNA that contains the target sequence.

 Primers: Short single-stranded DNA sequences complementary to target

regions.

 Forward primer (F) binds to one strand.

 Reverse primer (R) binds to the complementary strand.

 dNTPs: Building blocks (dATP, dTTP, dGTP, dCTP).

 DNA Polymerase: Enzyme that synthesizes new DNA strands.

Three steps of PCR:

 Denaturation (95°C): DNA strands separate.

 Annealing (50–65°C): Primers bind to complementary sequences.

 Extension (72°C): Taq polymerase synthesizes new DNA strands.

Factors affecting PCR:

Chemical Factors:

 Template length and type.

 Primer sequence (length, specificity, degeneracy).

 Buffer composition.

Physical Factors:

 Denaturation/annealing/extension times and temperatures.

 Number of cycles.

Primers are engineered for: is complementary to the target DNA

sequence.

 Leader sequence: Facilitates restriction enzyme cutting.

 Restriction site: For cloning purposes.

 Hybridization sequence: Complementary to the target DNA.

What is TAQ polymerase:

A thermostable DNA polymerase used in PCR.

PCR rationale: This primer will bind to the target DNA at the
hybridization region, be extended during PCR, and produce a product
that includes a restriction site with a leader sequence—ideal for
cloning into a vector.