Excel A-Level Biochemistry: Enzyme inhibition

Inhibitors are molecules that interact in some way with the enzyme to prevent it from working in its normal manner. There are a variety of types of inhibitors including: nonspecific, irreversible, reversible -- competitive and noncompetitive.

Michaelis-Menten Graphs

It is a plot made to record the initial velocity of an enzyme catalyzed reaction based on varying and increasing concentrations of substrate with a fixed quantity of enzyme. Only the initial rate is observed because if any other time point were observed there would be reverse reactions (since the reaction is typically on equilibrium).

If we have no substrate present, then there will be no reaction, so at [S] = 0, the rate will be 0. Adding substrate to the vessel will increase the amount of substrate binding to the enzyme and thus the reaction rate will increase. If we keep adding substrate, however, there will come a point when all the enzymes are working as fast as they can and are essentially saturated, therefore, the rate will not able to increase regardless of how much extra substrate is added. Thus, the plot of increasing substrate will be a curve that eventually flattens out reaching a maximum reaction rate, Vmax.

From the Michaelis-Menten equations, a constant (Km) is used which relates the equilibrium constants of the forward and reverse reactions all together. This point, where [S] = Km, has a rate exactly equal to Vmax/2. This Km value also describes precisely the effective dissociation of the substrate-enzyme complex. Thus, a high Km value implies the enzyme is not very good at holding onto its substrate whereas a low Km implies the enzyme has a very high affinity for its substrate. This value becomes critical in understanding how enzymes behave when inhibitors are added.

Lineweaver-Burk Double Reciprocal Plots

While the above graph is straight forward to understand, trying to determine exact points on a curve can be difficult. To avoid this, a second graph is often produced in which the reciprocals of the substrate values, [S] and reaction rates, V are plotted which produce a straight line that can be easily read. In this graph, Y-intercept is 1/Vmax and X-intercept is -1/Km, thus the important values can easily be extracted.

Inhibitor types

1. Competitive
   A competitive inhibitor is a molecule which directly competes with the enzyme's true substrate for binding to the active site. Thus everytime an inhibitor binds the active site, the enzyme will no longer be able to catalyze the reaction and the rate will decrease. If one is experimented with this type of inhibitor, increasing the concentration of the inhibitor will decrease the reaction rate, however, Vmax can still be achieved if enough substrate is added to cut compete the inhibitor. Because Km describes the dissociation constant for a substrate with its enzyme, adding an inhibitor will make it appear that the enzyme has a reduced affinity for the substrate
   
2. Noncompetitive
   The inhibitor binds at a site distinctly different from the active site (where the substrate normally binds). This alternative site on the enzyme is generally known as an allosteric site. Bind of the inhibitor at this site generally reduced the enzyme's ability to convert substrates into products at the active site (however the reverse can also be true, in which case it is an allosteric activator). When noncompetitive inhibitors are added to a reaction, Vmax reduces as the enzyme can no longer function as well as it could in the absence of the inhibitor. Note that the Km for the reaction does not change because while the enzyme isn't catalyzing the reaction as well, it can still bind the substrate just as well as it did before (Km = a measure of the affinity of the substrate to enzyme). Y-intercept increases (because Vmax in 1/Vmax is decreasing) but the 1/Km position stays the same.
   
3. Uncompetitive
   The inhibitor can only bind the enzyme-substrate complex, free enzyme is not a target of inhibition, but once a substrate enters so too can the inhibitor. Obviously, because enzymes which are bound to substrates can become blocked, the Vmax must be reduced. Km, however does not have as obvious a change. Because the enzyme-substrate normally exists in equilibrium with the enzyme and substrate, when you remove it from the equilibrium (by binding the inhibitor), the equilibrium shifts to replace the missing complex. As a consequence, more substrate binds to more free enzyme until a new equilibrium is established, When one observes this however, it appears as though the substrate has an increased affinity for enzyme (and thus a lower Km). The real reason for this effect however is simply because substrate has now been taken up to form substrate-enzyme and substrate-enzyme-inhibitor complexes, effectively consuming more substrate than the original, inhibitor-free equilibrium. The Y-intercept increases (because Vmax in 1/Vmax is decreasing) and X-intercept shifts to the left (because Km decreases thus -1/Km becomes larger and more negative). The slop will remain constant because both the Km and Vmax will change proportionally to each other and so the line will simply be observed to shift up and to the left.