Enzyme Inhibitors Biochemistry PDF

# Enzyme Inhibitors ## Introduction Enzymes can be inhibited by small molecules that bind either the active site or an allosteric site and thereby alter the reaction kinetics. Enzyme inhibition can act in a reversible (or dynamic) manner or in an irreversible manner. As the name implies, dynamic,...

# Enzyme Inhibitors ## Introduction Enzymes can be inhibited by small molecules that bind either the active site or an allosteric site and thereby alter the reaction kinetics. Enzyme inhibition can act in a reversible (or dynamic) manner or in an irreversible manner. As the name implies, dynamic, reversible inhibition can be undone once the molecule that caused it is removed. Reversible inhibitors are classified as competitive, uncompetitive, or mixed (with noncompetitive being a special case of mixed inhibition) based on their mechanism of action. In contrast, irreversible inhibition cannot be undone. This lesson describes the mechanisms of irreversible and reversible inhibition, and the graphs they produce are examined. ## 5.4.01 Irreversible Inhibitors Irreversible inhibition results from a permanent alteration to an enzyme that decreases that enzyme's activity. Typically, these alterations are the result of irreversible covalent modifications to the enzyme or from very tight (ie, low K_a) binding (Table 5.2). Because the modification is permanent, irreversible inhibition can only be overcome by synthesizing more unaltered enzyme; therefore, irreversible inhibition is energetically costly. Aside from protein degradation, irreversible inhibition is relatively uncommon as an endogenous (ie, native to the organism) means of regulation. Instead, irreversible inhibitors typically come from exogenous sources such as synthetic drugs or natural toxins. ## 5.4.02 Competitive Inhibitors Unlike irreversible inhibition, reversible inhibition does not cause a permanent change to the affected enzyme. Although some covalent regulatory mechanisms are dynamic and can be reversed (see Concept 6.2.02), this concept focuses on reversible regulators that inhibit their target enzyme through noncovalent means (ie, through binding-induced conformational changes). Broadly speaking, reversible inhibitors bind their target enzyme at one of two places: at the enzyme active site or at a different (ie, allosteric) site. The measured kinetics of the enzyme can differ based on where the inhibitor binds and whether the inhibitor binds free enzyme, the enzyme-substrate complex, or both. Consequently, reversible enzyme inhibitors are typically classified into one of several classes: competitive inhibitors, uncompetitive inhibitors, mixed inhibitors, and noncompetitive inhibitors. As the name implies, competitive inhibitors "compete" with the substrate for the free enzyme. Most commonly, this occurs if the competitive inhibitor is a substrate analog (ie, a molecule that structurally looks like, but is not, the substrate). Substrate analogs bind the enzyme active site but do not react. Importantly, an enzyme can bind only its substrate or a competitive inhibitor but cannot bind both at the same time, as shown in Figure 5.30. ## 5.4.03 Uncompetitive Inhibitors Unlike competitive inhibitors, which only bind free enzymes, uncompetitive inhibitors only bind the enzyme-substrate complex and thereby prevent the inhibitor-bound enzyme-substrate complex from completing its reaction. Inhibitor binding does not compete with substrate binding but neither is it completely independent of substrate binding; instead, uncompetitive inhibitor binding requires binding to the union of the enzyme and substrate together (Figure 5.33). ## 5.4.04 Mixed Inhibitors Whereas competitive inhibitors bind only free enzyme and uncompetitive inhibitors bind only the enzyme-substrate complex, mixed inhibitors can bind either free enzyme or the enzyme-substrate complex to prevent the reaction (Figure 5.35). ## 5.4.05 Noncompetitive Inhibitors A special subclass of mixed inhibitors binds the free enzyme and the enzyme-substrate complex with equal affinities (ie, $K_i$ = $K_i$'). As with other mixed inhibitors, $V_0$ is decreased at all substrate concentrations, including saturating substrate concentrations (ie, $V_{max}$ is decreased). However, because these inhibitors do not prefer either the free enzyme or the enzyme-substrate complex, the apparent $K_m$ value is neither increased nor decreased but remains unchanged by the inhibitor. Another way to think about this subclass of inhibitors is that substrate binding does not affect how well the inhibitor binds, and similarly inhibitor binding does not affect how well the substrate binds. The two events (ie, substrate binding and inhibitor binding) are completely independent. To denote this special property, a member of this subclass of mixed inhibitors is often called a purely noncompetitive inhibitor (Figure 5.37). ## Summary Competitive inhibitors are usually substrate analogs that bind at the enzyme's primary binding site (although some bind at allosteric sites). All other inhibitor classes must bind at an allosteric site. Whereas competitive inhibitors can be outcompeted (leaving $V_{max}$ unchanged), the ability of the other classes to bind the enzyme-substrate complex allows them to inhibit the enzyme even when the enzyme is saturated with substrate, which causes $V_{max}$ to decrease. The effect on apparent $K_m$ for each class depends on the relative affinity of each inhibitor for the free enzyme and the enzyme-substrate complex. The features of each inhibitor class are summarized in Table 5.5. | Type of Inhibition | Inhibitor Binds | Effect on $K_m$ | Effect on $V_{max}$ | |---|---|---|---| | Competitive | E only | Increases | No change | | Mixed | E more than ES | Increases | Decreases | | Noncompetitive (special case of mixed) |E and ES equally| No change | Decreases | | | ES more than E | Decreases | Decreases | | Uncompetitive| ES only | Decreases | Decreases by same factor as $V_{max}$ | * E = free enzyme; ES = enzyme-substrate complex. ## 5.4.06 Determining Inhibitor Types from Graphical Data The Michaelis-Menten plot was introduced in Concept 5.3.01 as a method of representing kinetic data by plotting $V_0$ on the y-axis against [S] on the x-axis. The Lineweaver-Burk plot introduced in Concept 5.3.02 is a linearization of the Michaelis-Menten plot that is particularly useful in interpreting changes in kinetic data, such as those caused by reversible inhibition. This concept explores the graphical changes in both Michaelis-Menten and Lineweaver-Burk plots brought about by inhibitors. ### Competitive Inhibitors As discussed in Concept 5.4.02, competitive inhibitors increase apparent $K_m$ and leave $V_{max}$ unchanged. On a Michaelis-Menten plot, this is represented as a right shift of $K_m$ with no change in the horizontal asymptote (ie, $V_{max}$). Because $V_0$ approaches (but never reaches) the $V_{max}$ value, an unchanged $V_{max}$ may be difficult to identify unless sufficiently high [S] values are tested, as shown in Figure 5.38 (left). Because the Lineweaver-Burk plot extrapolates the line of best fit beyond the collected data points, changes in the $K_m$ and $V_{max}$ values can be clearly identified by examining the x- and y-intercepts, respectively. The increase in $K_m$ changes the x-intercept (ie, -1/$K_m$) by shifting it rightward, toward the origin. Because $V_{max}$ is unchanged, the y-intercept (ie, 1/$V_{max}$) is unchanged. This results in an inhibited data curve that intersects the uninhibited data curve at the y-intercept. These changes are reflected in Figure 5.38 (right). ### Uncompetitive Inhibitors Uncompetitive inhibitors bind the unified enzyme-substrate complex and decrease both $K_m$ and $V_{max}$ by exactly the same factor. On a Michaelis-Menten plot, the horizontal asymptote representing the $V_{max}$ value is lowered, and $K_m$ is shifted to the left. An example of uncompetitive inhibition on a Michaelis-Menten plot is shown in Figure 5.39 (left). However, with simple visual inspection of a Michaelis-Menten plot, it can be difficult to discern between an uncompetitive inhibitor and an "uncompetitive-like" mixed inhibitor (which also decreases both $K_m$ and $V_{max}$ but by different factors). On a Lineweaver-Burk plot, decreases in the $K_m$ and $V_{max}$ values cause both the x- and y-intercepts, respectively, to shift away from the origin—more specifically, the x-intercept (ie, -1/$K_m$) shifts left and the y-intercept (ie, 1/$V_{max}$) shifts upward. Because both parameters shift by the same factor, the slope of the Lineweaver-Burk plot (ie, $K_m$/$V_{max}$) is unchanged, resulting in the inhibitor data producing a line that is parallel to the uninhibited data. An example of uncompetitive inhibition on a Lineweaver-Burk plot is shown in Figure 5.39 (right). ### Noncompetitive Inhibitors Noncompetitive inhibitors are a subclass of mixed inhibitors that cause a decrease in $V_{max}$ with no change to $K_m$. On a Michaelis-Menten plot, the horizontal asymptote representing the $V_{max}$ value is lowered and the $K_m$ is unchanged. An example of noncompetitive inhibition on a Michaelis-Menten plot is given in Figure 5.40 (left). However, because the reference $V_{max}$ value changes between the uninhibited and inhibited curves, pinpointing and comparing the $K_m$ values may be difficult unless they are explicitly given. On a Lineweaver-Burk plot, the decrease in $V_{max}$ causes the y-intercept (ie, 1/$V_{max}$) to shift away from the origin (more specifically, it shifts upward). In contrast, the x-intercept (ie, -1/$K_m$) is unchanged because $K_m$ is unchanged, resulting in an inhibited data curve that intersects the uninhibited curve at the x-intercept. A Lineweaver-Burk plot showing noncompetitive inhibition is shown in Figure 5.40 (right). ### Mixed Inhibitors Mixed inhibitors that are not purely noncompetitive come in two major subclasses: "competitive-like" mixed inhibitors, which increase the apparent $K_m$, and "uncompetitive-like" mixed inhibitors, which decrease the apparent $K_m$. In both subclasses, the $V_{max}$ decreases, so the horizontal asymptote that represents $V_{max}$ on a Michaelis-Menten plot also decreases. $K_m$ shifts to the right for "competitive-like" mixed inhibitors and to the left for "uncompetitive-like" mixed inhibitors. Example Michaelis-Menten plots are given in Table 5.6 (left). On a Lineweaver-Burk plot, the decrease in $V_{max}$ causes the y-intercepts (ie, 1/$V_{max}$) to shift away from the origin for both subclasses of mixed inhibitor-more specifically, they shift upward. For "competitive-like" mixed inhibitors, which increase the apparent $K_m$, the x-intercept shifts toward the origin (ie, to the right). For "uncompetitive-like" inhibitors, which decrease the apparent $K_m$, the x-intercept shifts away from the origin (ie, to the left). Note that the x-intercept cannot shift beyond the point where the inhibited and uninhibited curves are parallel. If the curves did become parallel, this would indicate true uncompetitive inhibition. Example Lineweaver-Burk plots depicting mixed inhibition are shown in Table 5.6 (right). Note that both the "competitive-like" and the "uncompetitive-like" mixed inhibitors cause an increase in the slopes of Lineweaver-Burk plots, indicating a reduction in apparent catalytic efficiency and resulting in nonparallel lines that intersect at points that are not on either axis. "Competitive-like" mixed inhibitors intersect at some point above the x-axis, whereas "uncompetitive-like" mixed inhibitors intersect below the x-axis.