Enzyme kinetics and inhibition

enzyme inhibitor development

nucleotide biosynthesis, cancer and chemotherapy

thymidylate synthase

methyl transfer from methylene THF to deoxyutacil monophosphate to generate DNA precursor deoxythymidine monophosphate

cofactor/coenzyme regeneration is essential for building DNA with thymidine triphosphate

substrate analogue 5-fluorouracil is used to treat

• Colorectal

• Pancreatic

• Breast

• Gastric

• Ovarian cancers

enzyme assays: continuous 

NADH exhibits a strong absorbance peak at 340 nm, while NAD+ has minimal absorbance at this wavelength.

enzyme assays: discontinuous measurements

Samples are taken at specific timepoints to monitor the reaction. The reaction is stopped before measurement.

HPLC (high performance liquid chromatography) - separation of products from samples and analyse.

mathematically describing enzyme catalysis

Most enzyme assays measure the consumption of the substrates or the formation of products over time

• At equilibrium the rate of product formation = the rate of product breakdown

The overall rate of change is 0. We know that the equilibrium position is dependent on ∆G of the reaction.

hyperbolic curve

 the reaction rate is dependent on substrate concentration and the closer to equilibrium the reaction is slower the rate of change: less substrate to react, more product

competitive inhibition

Binding of the inhibitor and substrate are mutually exclusive. The inhibitor binds to the active site and prevent substrate binding

non-competitive inhibition

Inhibition binds equally well to the free enzyme and ES complex. The inhibitor binds an allosteric site and prevents catalysis.

uncompetitive inhibition

Inhibition only binds to the ES complex to stop the chemical reaction

Lineweaver-Burk plot

Double reciprocal plots make visualisation of Vmax and Km changes with inhibition straightforward.

Double reciprocal plots are linear with intercepts proportional to Vmax and Km

Visualising different types of inhibition with Lineweaver-Berk plots of enzyme reactions. 

covalent inhibition

Reactive chemical groups form covalent bonds with the enzyme and irreversibly inhibit catalysis

• Penicillin antibiotic is a covalent irreversible inhibitor if transpeptidase involved in cell wall biosynthesis in bacteria. The antibiotic is bactericidal.

• Beta-lactam ring reacts with active site nucleophilic serine form a covalent bond which inhibits the enzyme and is highly stable.