CBS – L2 – Enzyme Properties & Kinetics

L/O: Review basics of enzymology: concept of catalysis, substrate specificity; effects of pH and temperature on the
rate of enzyme catalysed reaction.
Enzymes:
 Biological catalysts  Speed up the reaction rate.
 Does not alter equilibrium between reactants and products
 Activation energy  Decreases Gibbs free energy.
 Defined architecture.
 Substrate must have lower affinity for active site  To leave the enzyme.
 Structure:
 Complex 3D shape.
 1+ polypeptide chain  Stabilised by hydrogen bonds, electrostatic links & hydrophobic interactions.
 Sensitive to changes in environment!  Heat breaks weak bonds, and thus denatured.
 Active site.
 Functional groups  Stabilise the transition state of the reaction.
 Effect of temperature:
 Work best at 37 °C in humans.
 >37 °C  Heat inactivation.
 Effect of pH:
 Optimal pH depends on the target compartment.
 Outside  Denatured.
 Transition metals are utilised as cofactors for redox reactions.
Substrate specificity:
 Specificity  Determined by active site.
 Only the substrate of correct shape and charge fits.
 Compound compartmentalisation.
 Multiple enzymes which fully convert a substrate in a defined space.
 Group specificity.
 Enzyme catalyses 1 type of reaction.
 Stereo specificity.
 Enzyme acts only on 1 stereoisomer form.
Models:
 Lock and key:
 Substrates are complementary to the active site.
 Distortion of substrates allows them to be accommodated in the active site.
 Induced fit:
 Enzyme provides scaffold for the substrate to bind
 For example a phosphate group, thus locking on to complete the reaction.

Isozymes:
 Enzymes with different protein structures  Catalyse the same reaction.
 Coded for by different genes with distinct biochemical roles.
 Hexokinase (all cells), whereas glucokinase (liver).
 Catalyses glucose to G6P.
L/O: Describe how the rate of an enzyme reaction depends on the concentration of the substrate(s) and how this
is described by the Michaelis-Menten equation.
Enzyme kinetics “rate of an enzyme catalysed reaction”:
 Hyperbolic kinetics:
 Reaction rate is ∝ at ↓[S].
 Reaction rate is independent at ↑[S].

L/O: Define the terms: initial velocity, Km, Vmax and explain how these values may be determined
experimentally.
Definitions:
 Km  Michaelis constant.
 V0  Initial reaction velocity.
 Vmax  Maximal velocity of an enzyme.
 Kcat  Turnover number.
 Number of substrate molecules converted to product.
 Kcat/Km  Specificity constant.
 Used to compare catalytic efficiency between 2 enzymes
(Kcat/Km for each!).

L/O: Outline the clinical use of enzyme measurements.

Myocardial infarction:
 Differential diagnosis by investigating plasma levels of escaped enzymes.
 CK2 and LDH.
 LDH1/2  Increase in serum levels.

L/O: Explain with examples the terms reversible inhibition and irreversible inhibition.
Inhibition:
 Competitive inhibitors  Block the enzyme active site.
 Malonate  Inhibits succinate dehydrogenase.
 Non-competitive (reversible or irreversible)  Interfere in some other way with the catalytic mechanism.
 Reversible inhibition.
 Chealator (EDTA) can inhibit Mg2+-requiring enzymes.
 Irreversible inhibition.
 Organophosphorus inhibition of cholinesterase.
L/O: Explain the different effects of competitive, non-competitive inhibitors on enzyme kinetic parameters.

 Competitive inhibitor  Vmax same, Km is larger.

 Non-competitive inhibitor  Vmax is lower, Km is same.
L/O: Indicate with named examples, two clinical uses of enzymes inhibitors.
Heart failure:
 Treated using Captopril, an ACE inhibitor.
Alzheimer’s Disease:
 Treated using Donepezil, a reversible AChE inhibitor.
L/O: Summarise some of the important mechanisms for regulation of enzyme activity.
Allosteric regulation:
 Negative allosteric regulators  ATP and citrate on phosphofructokinase.
 Positive allosteric regulators  PEP and F16BP on pyruvate kinase.