Term 2 Lecture 6: Protein Structure And Function

Question 1

Protein structure and function is regulated by:

Answer 1

pH, heat, ligands (small molecules)
Consecutive reactions
feedback pathways
chemical modification by other enzymes and interactions with other proteins

Question 2

Physico chemical effects - pH and heat

Answer 2

Extremes of pH can denature proteins whilst small changes can regulate them

pH change affects charge on proteins/ ligands affecting ligand receptor interactions involving binding and catalysis

pH change can affect protein structure through effects of ionisation on as side chains

Function can change significantly due to denaturation e.g. in talin-actin binding protein involved in cell migration - changing pH affects ionisation of residues at top (pH sensor) causing confirmational changes to region at the bottom of the molecule affecting actin binding.

Small changes in pH affect biological molecules e.g. oxygen binding capacity of Hb

Question 3

Oxygen binding by Hb is pH dependent

Answer 3

This allows more to be loaded in the lungs and unloaded in the tissues - because lower pH is more efficient for unloading (due to loss of cooperativity)

High pO2 is required for loading.

Charge-charge interaction on salt bridge in deoxy haemoglobin inhibits oxygen binding (changes confirmation to T favouring)
At higher pH (6.6) histidine side chains are not protonated so salt bridge is not present and oxygen can bind (T form not favoured so R form occurs)

CO2+H20<-> H2OCO3 <-> HCO3- + H+

Reaction between CO2 & H2O catalysed by carbonic anhydrase, decrease in pH caused by dissociation of carbonic acid decreases affinity of Hb for O causing T form to be favoured

Question 4

O2 binding by Hb in lung/ in tissues

Answer 4

Lung/tissues

High O2/ low O2
Low CO2/ high CO2
Rel high pH/ rel low pH
R form favoured/ T form favoured
O2 bonds efficiently/released efficiently
Low levels of 2,3 DPG/ high levels
Hb not carbalymated/ Hb carbalymated

Question 5

pH affects enzymes and substrates

Answer 5

pH affects enzyme activity (Vmax) usually as a bell shaped curve with maximum as optimum pH

pH also affects substrate which can be part of what determines optimum pH

Stomach enzymes work best at low pH while intestine enzymes work best at slightly alkali pH (in humans)

Question 6

Effect of temperature on enzyme activity

Answer 6

Activity increases until the protein is denatured

Extremophiles such as thermophilic bacteria have very high optimum temp. E.g. 100⁰C and stable to >110⁰C for a short time. They are useful in industry e.g. Thermus aquaticus is the thermophilic bacteria from which Taq poly used in PCR was isolated.

Question 7

Regulation of protein binding by reversibly bound small molecules (ligands)

Answer 7

Binding of O to Hb is modulated by small molecule 2,3 biphosphoglycerate (2,3 BPG) an intermediate from the glycolytic pathway

2,3 BPG binds in central cavity of the Hb tetramer held by pos charged hist and lys side chains

2,3 BPG decreases affinity of Hb for O2 it preferentially binds to deoxy Hb stabilising T state (decreases affinity)

Intermediates in glycolytic pathway increase in level in cells under O2 limitation

High levels of 2,3 BPG in RBC under O2 stress aid O2 unloading where needed

Tissue under stress becomes acidified so this also has an effect.

Question 8

Enzymes : reversible/irreversible inhibitors

Answer 8

Reversible
Non-cov e.g. those forming ES complex can dissociate, restoring activity, common in metabolic regulation.
These usually resemble enzyme substrate/coenzyme or other metabolic intermediate

Irreversible
Form a cov bond between inhibitor and enzyme via a chemical reaction - when inactivated this way an enzyme usually can’t regain function.
These inhibitors are less common in metabolic regulation.
E.g. iodoacetamide - modifies enzyme

Question 9

Enzyme inhibition

Answer 9

Competitive:
Inhibitor binds to active site, prevents substrate from binding.
Can be countered by increasing conc. Of substrate to outcompete inhibitor.
The structure of the inhibitor is similar to that of the substrate

Non-competitive:
Inhibitor binds at a point that doesn’t prevent substrate binding. Binds to E & ES equally well. Effect cannot be countered by increase in substrate - effective for drugs.

Uncompetitive:
Affects substrate binding and turnover e.g. binds to an ES complex, when substrate binds to the enzyme it reveals a binding site for the inhibitor (induced fit)

Question 10

Michaelis Menten Vs Lineweaver burke plot to observe inhibition type

Answer 10

Michaelis Menten plots of enzyme inhibition constant Ki. Inhibitors change Vmax and Km →”apparent” Vmax & Km

Lineweaver burke plots are more informative w/distinct change according to inhibitor type

Competitive inhibition:
Km increased, Vmax unaffected

Uncompetitive inhibition:
Km reduced, Vmax reduced

Non-competitive inhibition (mixed inhib):
Km unaffected, V max reduced

See calculations in molecules and cells notebook 2

Question 11

Regulation by consecutive reaction/ feedback pathways

Answer 11

Basis of metabolism - coupling unfavourable w/favourable reactions

A favourable Gibbs free energy change (∆G <0) is exergonic
An unfavourable Gibbs free energy change (∆G >0) is endergonic

Endergonic & exergonic coupling is common in biochemistry

Two reactions can be directly coupled (same physical location, same time e.g. enzyme reaction) or indirectly coupled (energy from one reaction generates “potential energy” gradient which is used later in the system e.g. batteries.

Question 12

Direct coupling reaction example

Answer 12

Reaction 1:
Glucose + Pi → Glucose 6 phosphate
(∆G1)

Reaction 2:
ATP→ADP+Pi
(∆G2)

Reaction 3
Glucose + ATP
→ Glucose 6 phosphate+ADP
(∆G3=∆G1+∆G2)

Reaction 1 is thermodynamically unfavourable requiring a POS free energy change (endergonic) so does not happen by itself.

An enzyme (hexokinase) couples reaction 1 with reaction 2 the hydrolysis of ATP to ADP+Pi a reaction with a very negative free energy change (exergonic.)

This makes reaction 3, outcome is the addition of energy changes from reaction 1 & 2 added together. The overall exergonic outcome is a favoured reaction

Question 13

Consecutive coupling reaction example

Answer 13

Overall free energy change must be neg to be favourable.
In a multi step reaction with multiple barriers the slowest step has the largest barrier and is called the “rate limiting step’

Reaction profile for a simple consecutive reaction X→Y→Z rate constant K1 for first step and K2 for second step. So 2 activation barriers Ea1 & Ea2

1st step: K1>K2 (Ea1<Ea2)
X disappears quickly lots of Y made transiently
2nd step rate limiting

2nd step: K1=K2 (Ea1 = Ea2)
X disappears more slowly, some Y is made
Barriers equal

3rd step: K1<K2 ( Ea1>Ea2)
X disappears slowly little Y is made
First step rate limiting

Question 14

Role of non-competitive reversible inhibitors

Answer 14

Have a role in regulating enzyme activity in metabolic pathways - in feedback/forward pathways

A->B<->C<->D<->E<->F

Synthesis of cytidine triphosphate (TP a monomer in RNA)

First commited step A→B condensation of aspartame+ carbomylphosphate → N carbomyl-aspartate

Reaction catalysed by aspartate transcarbamoylase enzyme is inhibited by end product CTP

Enzyme shows non-michaelis Menten kinetics (sigmoid kinetics) due to interacting subunit binding sites causing cooperativity.

CTP binding stabilises T form of the enzyme decreasing affinity for substrates carbamoylphosphate and aspartame. CTP binds at a site on the enzyme separate from the active site where substrate binds (an example of feedback inhibition in metabolic pathway)

Question 15

Analysis of glucose combustion

Answer 15

If we assume jelly babies are pure glucose

C6H12O6 +6O2 →6CO2+H2O

One jelly baby ~22kcal (92kj) ~6g
1 mol glucose 180g so 30 jelly babies per mol & 1 mol = 2761 kjmol-¹

From standard tables at 298k ∆H combustion =2805kjmol-¹

∆H combustion is pretty close to manufacturers value (by bomb calorimetry accounting for sweet not being 100% Glucose)

Energy released ~20s ~138kjmol-¹s-¹
Or ~4.6kj baby -¹s-¹ (quite a lot)

Energy release is far better controlled in the body. The amount of energy released is the same whether the baby is burnt or metabolised

Question 16

Regulation by modification of proteins O2 binding by Hb also regulated by CO2 forming a cov bond w/ the protein

Answer 16

-CO2 decreases pH to aid O2 dissociation
-CO2 also has a direct effect by reacting with N terminal Val 1 residue of the Hb subunits polypeptide to form a carbonate
-although a cov bond is formed carbamylation is normally reversible
- effect of carbamate formation is to stabilise T form of Hb aiding O2 release
- cov modification regulates protein function
- serine →phosphoserine is catalysed by protein kinases. Addition of a neg phosphoryl group allows strong charge - charge interaction to drive changes in confirmation e.g. in SRC non-phosphorylated active form has open confirmation allowing substrates into kinase active site. Phosphorylated form is closed preventing access to active site - large movement in molecule binds serine tail region to SH2 region.

• dephosphorylation is catalysed by protein phosphates

Question 17

proteolytic cleavage is often involved in protein activation

Answer 17

-cleavage of specific peptide bonds in precursor form of a protein can lead to confirmational changes which activate the proteins functions
- irreversible activation is present in many biosystems where proteins must remain inactive until they reach their desired site of action
e.g. digestive proteases synthesised in the pancreas and activated where they are used in the gut
e.g. blood clotting cascade shows proteolytic activation from inactive precursors → enzymes resulting in macroscopic outcome

Question 18

Irreversible inhibitors form cov bonds with enzyme active sites

Answer 18

They react with functional groups in as side chains of the enzymes
E.g. iodoacetamide reacts with thiol groups
Unlike reversible inhibitors which must be present in excess to inactivate enzymes, irreversible inhibitors can react with enzymes on a 1:1 basis

E+i → EI

Useful as reagents and drugs.
Irreversible inhibitors can be made specific by making them resemble an enzymes normal substrate and making them chemically reactive with AA side chains in active site. Aka an ‘affinity label’
which can bond to a specific enzyme active site and form cov bonds
E.g. triose phosphate isomerase (TIM) +bromacetol phosphate (affinity label for TIM) → inactivated enzyme

Question 19

Gram neg and gram pos bacteria contain a layer of crosslinked peptidoglycan in their cell wall

Answer 19

• it’s essential for physical strength
• weakening of this layer leads to cell fragility and lysis due to osmotic shock
• the peptides contain some D-aa formation of bridge crosslinks involving a transpeptidase enzyme that catalyses incorporation of terminal D-ala & D-ala peptide.
• penicillin is a specific irreversible inhibitor that affects enzymes in bacterial cell wall synthesis and mimics D-Ala - D-ala C terminal part of the short peptide and bonds at the enzymes active site.
• penicillin reacts with side chain hydroxyl groups preventing catalytic enzyme activity (inactivates the enzyme)
• the reactive peptide bond in penicillin attached by serine hydroxyl group of enzyme active site forms a stable cov bond and enzyme is irreversibly inactivated preventing the bacteria from synthesising cell wall and causing the bacteria to die
  -however bacteria possess mechanisms allowing them to counteract wall weakening effects of antibiotics by developing resistance.

Question 20

Regulation by protein - protein interaction

Answer 20

Proteins can act as inhibitors by binding at the active site of an enzyme e.g. pancreatic trypsin inhibitor prevents any preactivated trypsin from digesting the pancreas.
It forms a tight binding complex, a residue on the loop of the inhibitor fits into enzyme active site, further non-cov reactions take place over surfaces of proteins in contact with it.
E.g. in the presence of Ca, Calmodulin binds to apoptosis associated “death” kinase initiating self destruction.

Other strategies for protein regulation:
- multiple forms of enzymes with different Km and Vmax values (isoenzymes)
E.g. lactate dehydrogenase has different forms in heart and skeletal muscle
- synthesis and degredation of proteins