3.8.1.1. Kinetics of Reversible Competitive Inhibition

• Competitive Inhibitor is an analog of the substrate, and binds to the active site of the enzyme. In virtually every case, competitive inhibitors bind in the same binding site as the substrate, but same-site binding is not a requirement. A competitive inhibitor could bind to an allosteric site of the free enzyme and prevent substrate binding, as long as it cannot bind to the allosteric site when the substrate is bound.

• Competitive inhibitors compete with the substrate for binding at the active site (as E + I).
• In the double reciprocal plot for a competitive inhibitor acting at the substrate site followings can be noticed: with increasing concentration of inhibitor, the V_max does not change; because the presence of the inhibitor can be overcome by higher substrate concentrations.
• However, the K_m of the substrate is increased (the K_d dissociation constant is apparently increased). This is because the concentration of substrate needed to reach V_max with an inhibitor is greater than the concentration of substrate needed to reach V_max without an inhibitor.Increasing value of K_m indicates a direct interaction of the inhibitor in the active site. The change in K_m (Michaelis-Menten constant) is parallel to the alteration in K_d.
• This also reflects the reversible nature of the inhibitor; there is always some concentration of substrate which can displace the inhibitor. Any given competitive inhibitor concentration can be overcome by increasing the substrate concentration in which case the substrate will outcompete the inhibitor in binding to the enzyme.

3.8.1.2. Kinetics of Reversible Non-Competitive Inhibition

• Non-competitive inhibitors combine with both the enzyme (E + I) and the enzyme-substrate (ES + I) complex. The inhibitor binds to a site other than the substrate site, and is thus independent of the presence or absence of substrate. This action results in a conformational change in the protein that affects a catalytic step and hence decreases or eliminates enzyme activity, i.e. formation of P.
• In the reciprocal plot, it is clear that a non-competitive inhibitor does not affect the binding of the substrate (K_m), but results in a decrease in V_max.  This can be explained by the fact that since inhibitor bound to an enzyme inactivates it, the more [EI] formed will lower [ES] and thus lower the overall rate of the reaction V_max.
• In the presence of a non-competitive inhibitor, the apparent enzyme affinity is equivalent to the actual affinity. In terms of Michaelis-Menten kinetics, K_m^app = K_m. This can be seen as a consequence of Le Chatelier's Principle because the inhibitor binds to both the enzyme and the enzyme-substrate complex equally so that the equilibrium is maintained. However, since some enzyme is always inhibited from converting the substrate to product, the effective enzyme concentration is lowered.
• When both the substrate and the inhibitor are bound, the enzyme-substrate-inhibitor complex cannot form product and can only be converted back to the enzyme-substrate complex or the enzyme-inhibitor complex. Non-competitive inhibition is distinguished from general mixed inhibition in that the inhibitor has an equal affinity for the enzyme and the enzyme-substrate complex.
• This type of inhibition reduces the maximum rate of a chemical reaction without changing the apparent binding affinity of the catalyst for the substrate (K_m^app). 6-hydroxyflavone is an example of Non-competitive inhibitors of CYP2C9 enzyme which bind in the allosteric binding site.