Inhibition of Enzymatic Reactions

Studies on Inhibitors are useful for:
1. Mechanistic studies to learn about how enzymes interact with their substrates.
2. Role of inhibitors in enzyme regulation.
3. Drugs if they inhibit aberrant biochemical reactions:
o Penicillin, Ampicillin, et al.: interfere with the synthesis of bacterial cell walls.
o Methotrexate: anti-cancer drug that affects DNA metabolism in actively growing cells
o Viagra: interferes with nitric oxide breakdown (NO is a vasodilator)
4. Understanding the role of biological toxins.
o Arsenate: mimics phosphate esters in enzyme reactions, but are easily hydrolyzed.
o Amino acid analogs: useful herbicides (i.e. roundup)
o Insecticides: chemicals targeted for the insect nervous system.

A. Competitive Inhibition
1. Inhibitor binds to the same site on the enzyme as the substrate, the active site.
2. Inhibitor only binds to the free enzyme.
3. Inhibitor is usually structurally very similar to the substrate. Examples:
o Succinate/Malonate
o ATP/AMP

The reaction scheme that corresponds to competitive inhibition is:

The inhibitor reduces the amount of E available for productive catalysis by the formation of the EI complex. The inhibitor does not affect the ES complex after it has formed. The dissociation constant for the inhibitor is KI = [E][I]/[EI].
There are two anticipated consequences of this additional competitive equilibrium:
1. Vmax is unchanged: At high levels of substrate all of the inhibitor is displaced by substrate.
2. KM is increased: Higher substrate concentrations are required to reach the maximal velocity.
Steady-state analysis of the effect of the inhibitor shows that KM is increased by a factor of (1 + [I]/KI). The resulting form of the Michaelis-Menten equation is:

Measurements of complete saturation curves (i.e. vo versus [S]) at different inhibitor concentrations can be used to obtain the dissociation constant for inhibitor binding. The Lineweaver-Burk plots will show an unchanged Vmax and a slope that increases with inhibitor concentration. (Figure 5.10). Campbell\'s equation (5.18) for the double reciprocal plot shows that the slope of the lines will be:

Thus, KI can be determined by plotting the slope values vs. [I]. The resulting secondary plot or \"replot\" will have a Y-axis intercept of KM/Vmax and a slope of KM/(VmaxKI). The value of KI is the intercept/slope of this replot.

B. Noncompetitive Inhibition:
In this case the inhibitor binds to both E and ES. Both the slope (KM/Vmax), and the Y-intercept (1/Vmax) of the Lineweaver-Burk plot increase The KI (\'s) are determined as above by replotting the slope and intercept values vs. [I].
1. Vmax is decreased: At high levels of substrate the inhibitor is still bound.
2. KM is increased: Higher [S] is required to reach the lower maximal velocity. (For \"simple noncompetitive inhibition\", KM is not changed, )
C. Uncompetitive Inhibition (not mentioned in Campbell)
1. The inhibitor binds directly to the ES complex.
2. The inhibitor does not have to bind at the active site.
3. The inhibitor does not have to resemble the substrate (e.g. an allosteric inhibitor).
Vmax is reduced: the amount of ESI formed depends on [I]. The solution to the steady-state equation results in the (1 + [I]/KI) factor multiplying the [S] term in the denominator of the Michaelis-Menten equation.
The slopes of the Lineweaver-Burk plot (KM/Vmax) are unchanged, but the Y-intercept increases by a factor of (1 + [I]/KI). The X-intercept shifts to the left by a factor (1 + [I]/KI).