Kinetic Tests for Determining Inhibition Mechanisms

The double-reciprocal plot (see Box 6-1) offers an easy way of determining whether an enzyme inhibitor is competitive, uncompetitive, or mixed. Two sets of rate experiments are carried out, with the enzyme concentration held constant in each set. In the first set, [S] is also held constant, permitting measurement of the effect of increasing inhibitor concentration [I] on the initial rate V0 (not shown). In the second set, [I] is held constant but [S] is varied. The results are plotted as 1/V0 versus 1/[S].

Figure 1 shows a set of double-reciprocal plots, one obtained in the absence of inhibitor and two at different concentrations of a competitive inhibitor. Increasing [I] results in a family of lines with a common intercept on the 1/V0 axis but with different slopes. Because the intercept on the 1/V0 axis equals 1/Vmax, we know that Vmax is unchanged by the presence of a competitive inhibitor. That is, regardless of the concentration of a competitive inhibitor, a sufficiently high substrate concentration will always displace the inhibitor from the enzyme's active site. Above the graph is the rearrangement of Equation 6-28 on which the plot is based. The value of a can be calculated from the change in slope at any given [I]. Knowing [I] and a, we can calculate KI from the expression

For uncompetitive and mixed inhibition, similar plots of rate data give the families of lines shown in Figures 2 and 3. Changes in axis intercepts signal changes in Vmax and Km.

FIGURE 2 Uncompetitive inhibition.

FIGURE 2 Uncompetitive inhibition.

TA medical therapy based on competition at the active site is used to treat patients who have ingested methanol, a solvent found in gas-line antifreeze. The liver enzyme alcohol dehydrogenase converts methanol to formaldehyde, which is damaging to many tissues. Blindness is a common result of methanol ingestion, because the eyes are particularly sensitive to formaldehyde. Ethanol competes effectively with methanol as an alternative substrate for alcohol dehydrogenase. The effect of ethanol is much like that of a competitive inhibitor, with the distinction that ethanol is also a substrate for alcohol dehydrogenase and its concentration will decrease over time as the enzyme converts it to acetaldehyde. The therapy for methanol poisoning is slow intravenous infusion of ethanol, at a rate that maintains a controlled concentration in the bloodstream for several hours. This slows the formation of formaldehyde, lessening the danger while the kidneys filter out the methanol to be excreted harmlessly in the urine. ■

Two other types of reversible inhibition, uncompet-itive and mixed, though often defined in terms of one-substrate enzymes, are in practice observed only with enzymes having two or more substrates. An uncompetitive inhibitor (Fig. 6-15b) binds at a site distinct from the substrate active site and, unlike a competitive inhibitor, binds only to the ES complex. In the presence of an uncompetitive inhibitor, the Michaelis-Menten equation is altered to where a' = 1 +

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  • hagosa mewael
    How to graph kinetic tests for determining inhibition mechanisms?
    7 years ago

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