In the previous section, (Introduction to Enzymes), the basics of enzymes and how they function was examined. In this section we will discuss how they are controlled. Recognizing how inhibitors change enzyme kinetics is often a very difficult topic to grasp however it is essential for answering enzyme questions on the MCAT.
Enzyme 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. The most common and generally examples of inhibitors are typically poisons and drugs which affect essential enzymes in the body.
While the mathematics of enzyme kinetics based off the Michaelis-Menten equations is not covered or examined, understanding their graphs is essential to understanding how enzymes can be inhibited and altered. The michaelis-menten graph 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 an equilibrium) and we do not these interfering with the observed rates.
Intuitively, we can guess what the graph will essentially look like. If we have no substrate present, then there will be no reaction, so at [substrate] = 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, the will come a point when all the enzymes are working as fast as they can and are essentially saturated, therefore, the rate will not be 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 (known as 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 Michaelis-Menten graphs is straightforward 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 produces a straight line that can be easily read. In this graph, the Y-intercept is equivalent to 1/Vmax and the X-intercept to -1/Km, thus the important values can easily be extracted.
You will not be expected to plot these graphs, however you must be able to determine how the intercepts of this graph change when inhibitors are introduced.
A competitive inhibitor is a molecule which directly competes with the enzyme's true substrate for binding to the active site. Thus every time 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 experimenting 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 out 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 (since some of the substrate is being kicked off for inhibitor instead) so the Km value will appear to increase (implying a greater dissociation).
A lineweaver-burk graph then, will shift where the x-intercept (1/Km) is, but the y-intercept (1/Vmax) will stay the same.
Noncompetitive inhibition occurs when 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 reduces the enzymes ability to convert substrates into products 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 (and Km is a measure of the affinity of the substrate to the enzyme).
When one observes noncompetitive inhibition in a lineweaver-burk plot the y-intercept increases (because Vmax in 1/Vmax is decreasing) but the 1/Km position stays the same.
Uncompetitive Inhibition occurs when an inhibitor can only bind the enzyme-substrate complex. That is, 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 actually 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 that the original, inhibitor-free equilibrium.
When one observes uncompetitive inhibitors in a lineweaver-burk plot, the y-intercept increases (because the Vmax in 1/Vmax is decreasing) and the x-intercept shifts to the left (because Km decreases and thus -1/Km becomes larger and more negative). In the most basic case, and the only conditions relevant to the MCAT), the slope will remain constant because both the Km and Vmax will be changing proportionally to each other and so the line will simply be observed to shift up and to the left.