Enzyme Kinetics
Part II. Enzyme Inhibition
II. Enzyme Inhibitors. A. Competitive Inhibition
Inhibitors of enzymes: Two types are considered - Competitive and Non-Competitive.
A Competitive Inhibitor has a chemical similarity to the substrate and competes with the substrate for binding to the active site of the enzyme. A good example to describe competitive inhibition is the mitochrondrial enzyme, succinate dehydrogenase:
Figure 2. (A) The reaction catalyzed by succinate dehydrogenase is the oxidation of succinate to fumarate. (B) Malonate and oxaloacetate are competitive inhibitors of succinate dehydrogenase.
Both these competitive inhibitors, malonate and oxaloacetate, look like succinate in their chemical character. Both inhibitors are dicarboxylic acids like the substrate succinate so they have groups which can bind in the same places in the active site of succinate dehydrogenase as the substrate. However, neither inhibitor has the capacity to undergo the reaction and so the enzyme is inhibited. Since these inhibitors simply bind to the enzyme, when the succinate concentration is high, they will be pushed out of the site by the substrate and the enzyme will catalyze the reaction as if no inhibitor were present.
An enzyme mechanism model of the action of a competitive inhibitor (Ic) based on the standard model of a Michaelis-Menten enzyme where E + S leads to the E-S complex, which leads to product P:
Figure 3A. Model of a Competitive Inhibitor (Ic) Interacting with the Enzyme (E) and an equation for the equilibrium formed between the Ic and E, which is governed by the inhibitor binding constant, Ki.
This model is the same as the one described in the previous lecture where enzyme (E) and substrate (S) bind to form the ES complex, which will go forward during catalysis to form product (P) and the free enzyme. In the presence of the competitive inhibitor, Ic, a complex forms with enzyme when the inhibitor binds, the E-Ic complex. This is a dead-end complex and can not go on to form product. However, the Ic is bound reversibly to the enzyme and when more substrate is added, the inhibition is overcome by pulling the enzyme free via the breakdown of the E-Ic complex, which is in equilibrium with free enzyme and free Ic. Another way to think about this is - when lots of substrate is added, the concentration of free enzyme (E) falls to such a low level, that some of the E-Ic complex must breakdown to replenish the free E demanded by the equilibrium between E and Ic. This can also be demonstrated by comparing the Vo versus [S] plots for uninhibited enzyme and enyzme in the presence of a competitive inhibitor:
Figure 3B. Vo versus [S] plot comparing the kinetics of the reaction in the absence of inhibitor and in the presence of the competitive inhibitor (Ic). At high [S], the initial velocity in the presence of Ic will be about the same as it is in the absence of the inhibitor. The concentration of S which will be required to overcome the effect of the competitive inhibitor will depend on the [Ic] (ie. concentration of the competitive inhibitor) and the Ki (ie. the binding constant of the inhibitor to enzyme).
In competitive inhibition, addition of more substrate will out compete the inhibitor and overcome the inhibition of the enzyme's catalytic rate - thus, the Vmax will be the same and only Km will be altered. This is most clearly illustrated with the double reciprocal plot comparing the uninhibited reaction to that in the presence of Ic.
Figure 4A. Double reciprocal plot for competitive inhibitor (Ic).
Here the uninhibted reaction gives the standard double reciprocal plot from which Km and Vmax can be calculated. The reaction in the presence of the competitive inhibitor yields apparent constants for the enzyme which are called the Km' and Vmax'. For the true competitive inhibitor, the Vmax' (apparent Vmax for inhibited enzyme) will be the same as the real Vmax, while the Km' (apparent Km for the inhibited enzyme) will be greater than the real Km. Thus, the -1/Km' will be smaller than -1/Km. After finding Km and Km', the Ki for the Ic can be calculated using the equation shown using the given concentration of the competitive inhibitor ([I]).
Figure 4B. Quantitative relationship between the Km' (apparent Km) and the real Km in the presence of a competitive inhibitor. This equation is used to calculate the Ki for the competitive inhibitor at known [Ic].
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©Wilbur H. Campbell, 1995; wcampbel@mtu.edu