Enzyme Kinetics in the Presence of an Inhibitor

Enzyme Kinetics in the Presence of an Inhibitor

What are Enzyme inhibitors?

Enzyme inhibitors are molecules that interact with an enzyme in various ways to prevent it from working properly. 1 They alter the activity of an enzyme by decreasing the enzyme's ability to bind substrate, lowering its catalytic turnover, or both. 2 The catalytic turnover is the maximum number of substrate molecules converted to product per enzyme molecule per unit of time, represented by this equation Kcat= Vmax/Et 3.

Did you know?

Almost all therapeutic drugs are enzyme inhibitors, from aspirin and penicillin to compounds used to treat HIV infection.4

This is a list of the drugs that are used to treat but not cure an HIV infection: Nucleoside/Nucleotide Reverse Transcriptase Inhibitors, Non-Nucleoside Reverse Transcriptase Inhibitors, protease inhibitors, and integrase inhibitors6. Notice how all of them work on an enzyme involved in the replication process of the viral nucleic acid.

Back to penicillin as an enzyme inhibitor; Penicillin kills susceptible bacteria by specifically inhibiting the transpeptidase that catalyzes the final step in cell wall biosynthesis, the cross-linking of peptidoglycan.7 The reaction is favoured by the strong resemblance between the peptide bond in the lactam ring of penicillin and the transition state complex of the natural transpeptidation reaction8. There are 2 types of inhibitors, reversible and irreversible, that are differentiated by the magnitude of affinity to their enzymes 9.

Penicillin vs normal substrate comparison 10

Irreversible inhibitors

They are also known as inactivators 12; since they bind tightly to an enzyme by covalent bonds and thus forming a permanent complex. An example of such inhibitor is penicillin.

Reversible inhibitors

They bind and dissociate with their enzyme in an equilibrium process10. They are not covalently bound to the enzyme and can dissociate at a significant rate. Reversible inhibitors are generally classified as competitive, noncompetitive, and uncompetitive with respect to their relationship to a substrate of the enzyme11.

1. COMPETITIVE INHIBITION

A competitive inhibitor "competes" with a substrate for binding at the enzyme's substrate recognition site and therefore is usually a close structural analog of the substrate 13; thus they lower the enzyme's likelihood of binding substrate and they slow the observed reaction velocity14.

Competitive inhibitors bind at the active site of the enzymes to form an E-I complex (Scheme 1). The inhibitor blocks the active site, and the substrate cannot bind until the inhibitor dissociates. Since the inhibitor and substrate compete for the same site, raising the substrate concentration can eventually overcome the inhibitor, and Vmax can be achieved. Although Vmax can be reached, a competitive inhibitor raises Km, indicating that the affinity of the enzyme for the substrate is lower in the presence of the inhibitor16.

Michaelis-Menten plot

Note: Ki is the dissociation constant for the EI complex19.

Lineweaver-Burk plot

The effect of a competitive inhibitor is both to move the x-intercept and increase the slope. Plots made with varying amounts of a competitive inhibitor will all cross at the same y-intercept17. Competitive inhibitors, therefore, increase the apparent Km of the enzyme (KM,app) because they raise the concentration of substrate necessary to saturate the enzyme. They have no effect on Vmax. 20

2. Non-competitive inhibitor or mixed inhibitors

This type of inhibitor does not compete

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