8.1 Enzymes Are Powerful and Highly Specific Catalysts

Most catalysts in biological systems are enzymes, and nearly all enzymes are proteins. Enzymes are highly specific and have great catalytic power. They can enhance reaction rates by factors of 10⁶ or more. Many enzymes require cofactors for activity. Such cofactors can be metal ions or small, vitamin-derived organic molecules called coenzymes.

8.2 Gibbs Free Energy Is a Useful Thermodynamic Function for Understanding Enzymes

Free energy (G) is the most valuable thermodynamic function for understanding the energetics of catalysis. A reaction can take place spontaneously only if the change in free energy (ΔG) is negative. The free-energy change of a reaction that takes place when reactants and products are at unit activity is called the standard free-energy change (ΔG°). Biochemists use ΔG°′, the standard free-energy change at pH 7. Enzymes do not alter reaction equilibria; rather, they increase the rate at which equilibrium is attained.

8.3 Enzymes Accelerate Reactions by Facilitating the Formation of the Transition State

Enzymes serve as catalysts by decreasing the free energy of activation of chemical reactions. Enzymes accelerate reactions by providing a reaction pathway in which the transition state (the highest-energy species) has a lower free energy and hence is more rapidly formed than in the uncatalyzed reaction.

The first step in catalysis is the formation of an enzyme–substrate complex. Substrates are bound to enzymes at active-site clefts from which water is largely excluded when the substrate is bound. The specificity of enzyme–substrate interactions arises mainly from hydrogen bonding, which is directional, and from the shape of the active site, which rejects molecules that do not have a sufficiently complementary shape. Enzymes facilitate formation of the transition state by a dynamic process in which the substrate binds to specific conformations of the enzyme, accompanied by conformational changes at active sites that result in catalysis.

8.4 The Michaelis–Menten Model Accounts for the Kinetic Properties of Many Enzymes

The kinetic properties of many enzymes are described by the Michaelis–Menten model. In this model, an enzyme (E) combines with a substrate (S) to form an enzyme–substrate (ES) complex, which can proceed to form a product (P) or to dissociate into E and S.

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The rate of formation of product V₀ is given by the Michaelis–Menten equation:

in which V_max is the reaction rate when the enzyme is fully saturated with substrate and K_M, the Michaelis constant, is the substrate concentration at which the reaction rate is half maximal. The maximal rate, V_max, is equal to the product of k₂, or k_cat, and the total concentration of enzyme. The kinetic constant k_cat called the turnover number, is the number of substrate molecules converted into product per unit time at a single catalytic site when the enzyme is fully saturated with substrate. Turnover numbers for most enzymes are between 1 and 10⁴ per second. The ratio of k_cat/K_M provides a measure of enzyme efficiency and specificity.

Allosteric enzymes constitute an important class of enzymes whose catalytic activity can be regulated. These enzymes, which do not conform to Michaelis–Menten kinetics, have multiple active sites. These active sites display cooperativity, as evidenced by a sigmoidal dependence of reaction velocity on substrate concentration.

8.5 Enzymes Can Be Inhibited by Specific Molecules

Specific small molecules or ions can inhibit even nonallosteric enzymes. In irreversible inhibition, the inhibitor is covalently linked to the enzyme or bound so tightly that its dissociation from the enzyme is very slow. Covalent inhibitors provide a means of mapping the enzyme’s active site. In contrast, reversible inhibition is characterized by a more rapid and less stable interaction between enzyme and inhibitor. A competitive inhibitor prevents the substrate from binding to the active site. It reduces the reaction velocity by diminishing the proportion of enzyme molecules that are bound to substrate. Competitive inhibition can be overcome by raising the substrate concentration. In uncompetitive inhibition, the inhibitor combines only with the enzyme–substrate complex. In noncompetitive inhibition, the inhibitor decreases the turnover number. Uncompetitive and noncompetitive inhibition cannot be overcome by raising the substrate concentration.

The essence of catalysis is selective stabilization of the transition state. Hence, an enzyme binds the transition state more tightly than it binds the substrate. Transition-state analogs are stable compounds that mimic key features of this highest-energy species. They are potent and specific inhibitors of enzymes. Proof that transition-state stabilization is a key aspect of enzyme activity comes from the generation of catalytic antibodies. Transition-state analogs are used as antigens, or immunogens, in generating catalytic antibodies.

8.6 Enzymes Can Be Studied One Molecule at a Time

Many enzymes are now being studied in singulo, at the level of a single molecule. Such studies are important because they yield information that is difficult to obtain in studies of populations of molecules. Single-molecule methods reveal a distribution of enzyme characteristics rather than an average value as is acquired with the use of ensemble methods.

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