Enzymology Flashcards
Enzymes
The OED defines an enzyme as a macromolecule that catalyses a biochemical reaction.
Thermodynamics vs Kinetics
- 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.
- It is therefore thermodynamically favoured, but so slow that it is not observed
Enzyme Bioenergetics
Enzymes reduce the activation energy required to allow a reaction to proceed.
This depends upon precise interactions, high specificity in molecular structure.
Structural Specificity: Protein Structure
- Hydrogen bonds
- Hydrophobic Interactions
- Cys-Cys Disulphide bond
- Ionic ‘Salt’ interactions
Phosphate-metal interactions
Stereoisomers
Stereoisomers: Same molecular and structural formula, differing in spatial arrangement of atoms
Enantiomers
Enantiomers (optical isomers) are stereoisomers which are mirror images - rotate the plane of polarised light opposite ways - commonly have a chiral (asymmetric) carbon.
Natural Amino Acids
Natural amino acids are L-enantiomers (except glycine, which is not chiral).
Hexokinase
Hexokinase:
ATP + Mg2+ glucose binding: induced fit forms transition state.
The shape of the ‘lock’ is not fixed.
Michaelis-Menten Enzyme Kinetics
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
Effects of pH on Enzyme Activity
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.
Effects of Temperature on Enzyme Activity
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.
Glucose Metabolism
Glycolysis -> PDH (Pyruvate Dehydrogenase)-> TCA (Tricarboxylic Acid ) cycle-> Oxidative Phosphorylation
Glycolysis
Multistep conversion of glucose to pyruvate
Metabolic regulation is complex, but key regulatory steps are catalysed by hexokinase (HK) and phosphofructokinase (PFK).
Phosphofructokinase (PFK)
Relatively far from thermodynamic equilibrium
Responsive to substrate concentration (not saturated)
Important allosteric effectors
A site of feedback control
Allostery
Allosteric regulation is the regulation of an enzyme by binding an effector molecule at a site other than the active site
Allosteric Regulation in PFK
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.
Multisite Control of Glycolysis
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.
Classes of Competitive Inhibition
Competitive Inhibitors
Non-competitive Inhibitors
Uncompetitive inhibitors
Competitive Inhibitors
- 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’
Non-competitive Inhibitors
- 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
Uncompetitive Inhibitors
- Binds to enzyme-substrate complex
Decreases Km and Vmax
Acetylcholinesterase in neurotransmission
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.
Cooperativity
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
