Enzyme Kinetics as an Approach to Understanding Mechanism: -Enzymes Are Subject to Reversible or Irreversible Inhibition

Enzyme inhibitors are molecular agents that interfere with catalysis, slowing or halting enzymatic reactions. Enzymes catalyze virtually all cellular processes, so it should not be surprising that enzyme inhibitors are among the most important pharmaceutical agents known. For example, aspirin (acetylsalicylate) inhibits the enzyme that catalyzes the first step in the synthesis of prostaglandins, compounds involved in many processes, including some that produce pain. The study of enzyme inhibitors also has provided valuable information about enzyme mechanisms and has helped de fine some metabolic pathways. There are two broad classes of enzyme inhibitors: reversible and irreversible.

Reversible Inhibition One common type of reversible inhibitionis called competitive (Fig. 6–15a). A competitive inhibitorcompetes with the substrate for the active site of an enzyme. While the inhibitor (I) occupies the active site it prevents binding of the substrate to the enzyme. Many competitive inhibitors are com pounds that resemble the substrate and combine with the enzyme to form an EI complex, but without leading to catalysis. Even fleeting combinations of this type will reduce the efficiency of the enzyme. By taking into account the molecular geometry of inhibitors that resemble the substrate, we can reach conclusions about which parts of the normal substrate bind to the enzyme. Competitive inhibition can be analyzed quantitatively by steady-state kinetics. In the presence of a competitive inhibitor, the Michaelis-Menten equation (Eqn 6–9) becomes

Where

Equation 6–28 describes the important features of competitive inhibition. The experimentally determined variable αKm, the Km observed in the presence of the inhibitor, is often called the “apparent” Km. Because the inhibitor binds reversibly to the enzyme, the competition can be biased to favor the substrate simply by adding more substrate. When [S] far exceeds [I], the probability that an inhibitor molecule will bind to the enzyme is minimized and the reaction exhibits a normal V_max. However, the [S] at which V0=1/2 Vmax, the apparent Km, increases in the presence of inhibitor by the factor α. This effect on apparent K_m, combined with the absence of an effect on V_max, is diagnostic of competitive inhibition and is readily revealed in a double reciprocal plot (Box 6–2). The equilibrium constant for inhibitor binding, KI, can be obtained from the same plot.

FIGURE 6–15 Three types of reversible inhibition. (a)Competitive inhibitors bind to the enzyme’s active site. (b)Uncompetitive inhibitors bind at a separate site, but bind only to the ES complex. KI is the equilibrium constant for inhibitor binding to E; KI is the equilibrium con stant for inhibitor binding to ES. (c)Mixed inhibitors bind at a separate site, but may bind to either E or ES.

A 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. Blind ness 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, uncompetitive 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

As described by Equation 6–29, at high concentrations of substrate, V0 approaches V_max/α. Thus, an uncompetitive inhibitor lowers the measured Vmax. Apparent Km also decreases, because the [S] required to reach one-half Vmax decreases by the factor α. A mixed inhibitor (Fig. 6–15c) also binds at a site distinct from the substrate active site, but it binds to ei ther E or ES. The rate equation describing mixed inhibition is

where and are defined as above. A mixed inhibitor usually affects both K_m and V_max. The special case of α َ=α, rarely encountered in experiments, classically has been defined as noncompetitive inhibition. Ex amine Equation 6–30 to see why a noncompetitive inhibitor would affect the Vmax but not the Km.

Equation 6–30 serves as a general expression for the effects of reversible inhibitors, simplifying to the expressions for competitive and uncompetitive inhibition when α َ=1.0 or α=1.0 respectively. From this expression we can summarize the effects of inhibitors on individual kinetic parameters. For all reversible inhibitors, apparent Vmax Vmax/ α  َ, because the right side of Equation 6–30 always simplifies to Vmax/ at sufficiently high substrate concentrations. For competitive inhibitors, α َ=1.0 and can thus be ignored. Taking this expression for apparent Vmax, we can also derive a general expression for apparent Km to show how this parameter changes in the presence of reversible inhibitors. Apparent K_m, as always, equals the [S] at which V₀ is one-half apparent Vmax or, more generally, when V₀=Vmax/2 α َ. This condition is met when [S]= α km/ α َ This expression is simpler when either α or α َ. is 1.0 (for uncompetitive or competitive inhibitors), as summarized in Table 6–9.

In practice, uncompetitive and mixed inhibition are observed only for enzymes with two or more substrates—say, S1 and S2—and are very important in the experimental analysis of such enzymes. If an inhibitor binds to the site normally occupied by S1, it may act as a competitive inhibitor in experiments in which [S1] is varied. If an inhibitor binds to the site normally occupied by S2, it may act as a mixed or uncompetitive inhibitor of S1. The actual inhibition patterns observed depend on whether the S1- and S2-binding events are ordered or random, and thus the order in which substrates bind and products leave the active site can be determined. Use of one of the reaction products as an inhibitor is often particularly informative. If only one of two reaction products is present, no reverse reaction can take place. However, a product generally binds to some part of the active site, thus serving as an inhibitor. Enzymologists can use elaborate kinetic studies involving different combinations and amounts of products and inhibitors to develop a detailed picture of the mechanism of a bisubstrate reaction.

Irreversible Inhibition The irreversible inhibitors are those that bind covalently with or destroy a functional group on an enzyme that is essential for the enzyme’s activity, or those that form a particularly stable noncovalent association. Formation of a covalent link between an irreversible inhibitor and an enzyme is common. Ir reversible inhibitors are another useful tool for studying reaction mechanisms. Amino acids with key catalytic functions in the active site can sometimes be identified by determining which residue is covalently linked to an inhibitor after the enzyme is inactivated. An example is shown in Figure 6–16. A special class of irreversible inhibitors is the suicide inactivators. These compounds are relatively un reactive until they bind to the active site of a specific enzyme. A suicide inactivator undergoes the first few chemical steps of the normal enzymatic reaction, but instead of being transformed into the normal product, the inactivator is converted to a very reactive compound that combines irreversibly with the enzyme. These com pounds are also called mechanism-based inactive tors, because they hijack the normal enzyme reaction mechanism to inactivate the enzyme. Suicide inactivators play a significant role in rational drug design, a modern approach to obtaining new pharmaceutical agents in which chemists synthesize novel substrates based on knowledge of substrates and reaction mechanisms. A well-designed suicide inactivator is specific for a single enzyme and is unreactive until within that en zyme’s active site, so drugs based on this approach can offer the important advantage of few side effects.

FIGURE 6–17 The pH-activity profiles of two enzymes. These curves are constructed from measurements of initial velocities when the re action is carried out in buffers of different pH. Because pH is a logarithmic scale reflecting tenfold changes in [H+], the changes in V0are also plotted on a logarithmic scale. The pH optimum for the activity of an enzyme is generally close to the pH of the environment in which the enzyme is normally found. (a)Pepsin, which hydrolyzes certain peptide bonds of proteins during digestion in the stomach, has a pH optimum of about 1.6. The pH of gastric juice is between 1 and 2. (b)Glucose 6-phosphatase of hepatocytes (liver cells), with a pH optimum of about 7.8, is responsible for releasing glucose into the blood. The normal pH of the cytosol of hepatocytes is about 7.2.

side chains can be significantly altered. For example, a nearby positive charge can lower the pKa of a Lys residue, and a nearby negative charge can increase it. Such effects sometimes result in a pKa that is shifted by several pH units from its value in the free amino acid. In the enzyme acetoacetate decarboxylase, for example, one Lys residue has a pKa of 6.6 (compared with 10.5 in free lysine) due to electrostatic effects of nearby positive charges.

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ضمن برنامج (سراج المنتظرين).. المجمع العلمي يكرم الطلبة الحاصلين على الإعفاء العام في المثنى

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ثورة في علاج الصمم.. علاج جيني يحقق نتائج استثنائية في استعادة السمع

منهج جديد باستخدام الساعات الذرية يعيد طرح سؤال: ما طبيعة الزمن؟

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Enzyme Kinetics as an Approach to Understanding Mechanism: -Pre–Steady State Kinetics Can Provide Evidence for Specific Reaction Steps

Enzyme Kinetics as an Approach to Understanding Mechanism:- Many Enzymes Catalyze Reactions with Two or More Substrates

Enzyme Kinetics as an Approach to Understanding Mechanism: -Kinetic Parameters Are Used to Compare Enzyme Activities

Enzyme Kinetics as an Approach to Understanding Mechanism: -The Relationship between Substrate Concentration and Reaction Rate Can Be Expressed Quantitatively

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How Enzymes Work:- Binding Energy Contributes to Reaction Specificity and Catalysis

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Protein Interactions Modulated by Chemical Energy: Actin, Myosin, and Molecular Motors: -Additional Proteins Organize the Thin and Thick Filaments into Ordered Structures