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Chapter: Biochemistry: The Behavior of Proteins: Enzymes

Enzyme Inhibition

How can we identify a competitive inhibitor?

Enzyme Inhibition

An inhibitor, as the name implies, is a substance that interferes with the action of an enzyme and slows the rate of a reaction. A good deal of information about enzymatic reactions can be obtained by observing the changes in the reaction caused by the presence of inhibitors. Inhibitors can affect an enzymatic reaction in two ways. A reversible inhibitor can bind to the enzyme and subsequently be released, leaving the enzyme in its original condition. An irreversible inhibitor reacts with the enzyme to produce a protein that is not enzymatically active and from which the original enzyme cannot be regenerated.

Two major classes of reversible inhibitors can be distinguished on the basis of the sites on the enzyme to which they bind. One class consists of compounds very similar in structure to the substrate. In this case, the inhibitor can bind to the active site and block the substrateÕs access to it. This mode of action is called competitive inhibition because the inhibitor competes with the substrate for the active site on the enzyme. Another major class of reversible inhibitors includes any inhibitor that binds to the enzyme at a site other than the active site and, as a result of binding, causes a change in the structure of the enzyme, especially around the active site. The substrate is still able to bind to the active site, but the enzyme cannot catalyze the reaction when the inhibitor is bound to it. This mode of action is called noncompetitive inhibition (Figure 6.11).


The two kinds of inhibition can be distinguished from one another in the laboratory. The reaction is carried out in the presence of inhibitor at several substrate concentrations, and the rates obtained are compared with those of the uninhibited reaction. The differences in the Lineweaver-Burk plots for the inhibited and uninhibited reactions provide the basis for the comparison.

How can we identify a competitive inhibitor?

In the presence of a competitive inhibitor, the slope of the Lineweaver-Burk plot changes, but the y intercept does not. (The x intercept also changes.) Vmax is unchanged, but KM increases. More substrate is needed to get to a given rate in the presence of inhibitor than in its absence. This point speciÞcally applies to the speciÞc value Vmax/2 (recall that at Vmax/2, the substrate concentration, [S], equals KM) (Figure 6.12). Competitive inhibition can be overcome by a sufÞciently high substrate concentration.



In the presence of a competitive inhibitor, the equation for an enzymatic reaction becomes


where EI is the enzyme-inhibitor complex. The dissociation constant for the enzyme-inhibitor complex can be written


It can be shown algebraically (although we shall not do so here) that, in the presence of inhibitor the value of KM increases by the factor


If we substitute KM (1 + [I]/KI) for KM in Equation 6.17, we obtain


Here the term 1/V takes the place of the y coordinate, and the term 1/[S] takes the place of the x coordinate, as was the case in Equation 6.17. The intercept 1/Vmax, the b term in the equation for a straight line, has not changed from the earlier equation, but the slope KM/Vmax in Equation 6.17 has increased by the fac-tor (1 + [I]/KI). The slope, the m term in the equation for a straight line, is now


accounting for the changes in the slope of the Lineweaver-Burk plot. Note that the y intercept does not change. This algebraic treatment of competitive inhibition agrees with experimental results, validating the model, just as experimental results validate the underlying Michaelis-Menten model for enzyme action. It is important to remember that the most distinguishing characteristic of a competitive inhibitor is that substrate or inhibitor can bind the enzyme, but not both. Because both are vying for the same location, sufÞciently high substrate will ÒoutcompeteÓ the inhibitor. This is why Vmax does not change; it is a measure of the velocity at inÞnite [substrate].

How can we identify a noncompetitive inhibitor?

The kinetic results of noncompetitive inhibition differ from those of competitive inhibition. The Lineweaver-Burk plots for a reaction in the presence and absence of a noncompetitive inhibitor show that both the slope and the y intercept change for the inhibited reaction (Figure 6.13), without changing the x intercept. The value of Vmax decreases, but that of KM remains the same; the inhibitor does not interfere with the binding of substrate to the active site. Increasing the substrate concentration cannot overcome noncompetitive inhibition because the inhibitor and substrate are not competing for the same site.



The reaction pathway has become considerably more complicated, and sev-eral equilibria must be considered.


In the presence of a noncompetitive inhibitor, I, the maximum velocity of the reaction, VImax, has the form (we shall not do the derivation here)


where KI is again the dissociation constant for the enzyme-inhibitor complex, EI. Recall that the maximum rate, Vmax, appears in the expressions for both the slope and the intercept in the equation for the Lineweaver-Burk plot (Equation 6.17):


In noncompetitive inhibition, we replace the term Vmax with the expression for V Imax, to obtain


The expressions for both the slope and the intercept in the equation for a Lineweaver-Burk plot of an uninhibited reaction have been replaced by more complicated expressions in the equation that describes noncompetitive inhibition. This interpretation is borne out by the observed results. With a pure, noncompetitive inhibitor, the binding of substrate does not affect the binding of inhibitor, and vice versa. Because the KM is a measure of the afÞnity of the enzyme and substrate, and because the inhibitor does not affect the binding, the KM does not change with noncompetitive inhibition.

The two types of inhibition presented here are the two extreme cases. There are many other types of inhibition. Uncompetitive inhibition is seen when an inhibitor can bind to the ES complex but not to free E. A Lineweaver-Burk plot of an uncompetitive inhibitor shows parallel lines. The Vmax decreases and the apparent KM decreases as well. Noncompetitive inhibition is actually a limiting case of a more general inhibition type called mixed inhibition. With a mixed inhibitor, the same binding diagram is seen as in the preceding equilibrium equations but, in this case, the binding of inhibitor does affect the binding of substrate and vice versa. A Lineweaver-Burk plot of an enzyme plus mixed inhibitor gives lines that intersect in the left-hand quadrant of the graph. The KMincreases, and the Vmaxdecreases.



Summary

Inhibitors are compounds that bind to enzymes and reduce the rate of catalysis.

Two principal types of inhibitors are competitive and noncompetitive.

Competitive inhibitors bind to the active site of an enzyme and prevent the simultaneous binding of substrate.

Noncompetitive inhibitors bind to enzymes at a site other than the active site, but they alter the active site in such a way to reduce the catalytic effi-ciency of the enzyme.

The type of inhibition can be determined by using a Lineweaver-Burk plot.

 

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