Enzymology

Question 1

Enzymes

Answer 1

The OED defines an enzyme as a macromolecule that catalyses a biochemical reaction.

Question 2

Thermodynamics vs Kinetics

Answer 2

• The reaction C(s) diamond –> C(s) Graphite has a negative change in Gibbs Free Energy at 25 degrees at 1atm
  
  • It is therefore thermodynamically favoured, but so slow that it is not observed
    Thermodynamics does not determine kinetics.

Question 3

Enzyme Bioenergetics

Answer 3

Enzymes reduce the activation energy required to allow a reaction to proceed.
This depends upon precise interactions, high specificity in molecular structure.

Question 4

Structural Specificity: Protein Structure

Answer 4

1. Hydrogen bonds
   
   2. Hydrophobic Interactions
   3. Cys-Cys Disulphide bond
   4. Ionic ‘Salt’ interactions
      Phosphate-metal interactions

Question 5

Stereoisomers

Answer 5

Stereoisomers: Same molecular and structural formula, differing in spatial arrangement of atoms

Question 6

Enantiomers

Answer 6

Enantiomers (optical isomers) are stereoisomers which are mirror images - rotate the plane of polarised light opposite ways - commonly have a chiral (asymmetric) carbon.

Question 7

Natural Amino Acids

Answer 7

Natural amino acids are L-enantiomers (except glycine, which is not chiral).

Question 8

Hexokinase

Answer 8

Hexokinase:
ATP + Mg2+ glucose binding: induced fit forms transition state.
The shape of the ‘lock’ is not fixed.

Question 9

Michaelis-Menten Enzyme Kinetics

Answer 9

Michaelis-Menten Enzyme Kinetics:
Reaction rate (V) as a function of substrate (S) concentration.
Simplest case
‘Hyperbolic’ curve
Two parameters:
Vmax = maximal rate
Km = [S] for half-maximal rate

Question 10

Effects of pH on Enzyme Activity

Answer 10

pH effects on amino acid ionization influence 3D protein structure. Affects ionic ‘salt’ interactions as pH affects charges of the cations/anions of the amino acids.

Question 11

Effects of Temperature on Enzyme Activity

Answer 11

Higher Temperature = greater kinetic energy of reactants (Arrhenius effect)
Higher temperature eventually denatures enzyme protein as hydrogen bonds break in the tertiary structure of the enzyme.
Note: Polymerase Chain Reaction (PCR) depends on thermostable DNA polymerase from extremophile bacteria.

Question 12

Glucose Metabolism

Answer 12

Glycolysis -> PDH (Pyruvate Dehydrogenase)-> TCA (Tricarboxylic Acid ) cycle-> Oxidative Phosphorylation

Question 13

Glycolysis

Answer 13

Multistep conversion of glucose to pyruvate
Metabolic regulation is complex, but key regulatory steps are catalysed by hexokinase (HK) and phosphofructokinase (PFK).

Question 14

Phosphofructokinase (PFK)

Answer 14

Relatively far from thermodynamic equilibrium
Responsive to substrate concentration (not saturated)
Important allosteric effectors
A site of feedback control

Question 15

Allostery

Answer 15

Allosteric regulation is the regulation of an enzyme by binding an effector molecule at a site other than the active site

Question 16

Allosteric Regulation in PFK

Answer 16

Basic negative feedback mechanism:
PFK is stimulated by fall in [ATP] and rise in [AMP]
This increases ATP production
Acidification accompanying lactate production limits this
But metabolic control is more complicated than effects on single, key enzymes.

Question 17

Multisite Control of Glycolysis

Answer 17

Theory suggests and experiment confirms that control of flux is distributed over several enzymes
In e.g. cancer cells, which are heavily glycolytic (the Warburg effect) glycolysis responds mainly to glucose transport (GLUT), hexokinase (HK), phosphofructokinase (PFK) and lactate efflux (via monocarboxylate transporters MCT). The enzymes of the lower part of glycolysis have little effect on flux.

Question 18

Classes of Competitive Inhibition

Answer 18

Competitive Inhibitors
Non-competitive Inhibitors
Uncompetitive inhibitors

Question 19

Competitive Inhibitors

Answer 19

• Compete with the substrate at active site of free enzyme
  
  • Raises Km, no effect on Vmax (can be ‘competed’ out by substrate
  • Noncovalent, reversible
    e.g. many drugs’

Question 20

Non-competitive Inhibitors

Answer 20

• Alters enzyme activity by binding of allosteric site
  
  • Lowers Vmax, no effect on Km
  • May be irreversible
    e.g. many poisons: CN, CO, heavy metals

Question 21

Uncompetitive Inhibitors

Answer 21

• Binds to enzyme-substrate complex
  Decreases Km and Vmax

Question 22

Acetylcholinesterase in neurotransmission

Answer 22

Acetylcholine esterase (AChE) inhibitors increase synaptic Ach concentration and are used to enhance neurotransmission e.g. Alzheimer’s
Because of potential toxicity, competitive inhibitors can be titrated are preferable to non-competitive inhibitors.

Question 23

Cooperativity

Answer 23

This quaternary structure of Hb means interaction between the binding of successive oxygen molecules to the haem groups.
Steep sections of sigmoid O2 dissociation curve means big change in O2 content for small change in pO2