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8885dc06_190-2371/27/047:13 AM Page200mac76mac76:385 Chapter 6 Enzymes Reaction Ra overall catalytic mechanism. Once a substrate is bound enhancement to an enzyme, properly positioned catalytic functional groups aid in the cleavage and formation of bonds by a CHa-C-OR CHs variety of mechanisms, including general acid-base catalysis, covalent catalysis, and metal ion catalysis CHs-C These are distinct from mechanisms based on binding energy, because they generally involve transient cova- CH3-C-0- lent interaction with a substrate or group transfer to or from a substrate (b) C-OR General Acid-Base Catalysis Many biochemical reactions involve the formation of unstable charged intermedi k(s-1) ates that tend to break down rapidly to their con stituent reactant species, thus impeding the reaction (Fig. 6-8). Charged intermediates can often be stabi- lized by the transfer of protons to or from the substrate (c) or intermediate to form a species that breaks down -OR more readily to products. For nonenzymatic reactions k(s-1) 10M the proton transfers can involve either the constituents of water alone or other weak proton donors or accep- tors. Catalysis of this type that uses only the (HsO or OH ions present in water is referred to as FIGURE 6-7 Rate enhancement by entropy reduction Shown here specific acid-base catalysis. If protons are trans are reactions of an ester with a carboxylate group to form an anhy ferred between the intermediate and water faster than dride. The R group is the same in each case. (a) For this bimolecular the intermediate breaks down to reactants the inter- reaction, the rate constant k is second order, with units of m-Is mediate is effectively stabilized every time it forms. No (b)When the two reacting groups are in a single molecule, the reac. additional catalysis mediated by other proton accep- tion is much faster. For this unimolecular reaction k has units of tors or donors will occur. In many cases, however, Dividing the rate constant for (b) by the rate constant for (a) gives a water is not enough. The term general acid-base rate enhancement of about 105M. (The enhancement has units of mo. catalysis refers to proton transfers mediated by other larity because we are comparing a unimolecular and a bimolecular classes of molecules. For nonenzymatic reactions in reaction)Put another way, if the reactant in(b)were present at a con. aqueous solutions, this occurs only when the unstable centration of 1 M, the reacting groups would behave as though they reaction intermediate breaks down to reactants faste were present at a concentration of 10 M. Note that the reactant in(b) than protons can be transferred to or from water. Many has freedom of rotation about three bonds (shown with curved weak organic acids can supplement water as proton rows), but this still represents a substantial reduction of entropy over donors in this situation, or weak organic bases can (a). If the bonds that rotate in(b)are constrained as in(c), the en- serve as proton acceptors tropy is reduced further and the reaction exhibits a rate enhancement In the active site of an enzyme a number of amino of10° M relative to(a) acid side chains can similarly act as proton donors and acceptors(Fig. 6-9). These groups can be precisely po- sitioned in an enzyme active site to allow proton trans This is referred to as induced fit, a mechanism postu- fers, providing rate enhancements of the order of 10to lated by Daniel Koshland in 1958. Induced fit serves to 10. This type of catalysis occurs on th e vast majority bring specific functional groups on the enzyme into the of enzymes. In fact, proton transfers are the most com- proper position to catalyze the reaction. The conforma- mon biochemical reactions tional change also permits formation of additional weak bonding interactions in the transition state. In either case, Covalent Catalysis In covalent catalysis, a transient co- the new enzyme conformation has enhanced catalytic valent bond is formed between the enzyme and the sub- properties. As we have seen, induced fit is a common fea- strate. Consider the hydrolysis of a bond between ture of the reversible binding of ligands to proteins(Chap- groups A and B ter 5) Induced fit is also important in the interaction of almost every enzyme with its substrate. A-B a+B Specific Catalytic Groups Contribute to Catalysis In the presence of a covalent catalyst(an enzyme with a nucleophilic group X: the reaction becomes In most enzymes, the binding energy used to form the ES complex is just one of several contributors to the A-B+X A-X+B-A+X:+BThis is referred to as induced fit, a mechanism postulated by Daniel Koshland in 1958. Induced fit serves to bring specific functional groups on the enzyme into the proper position to catalyze the reaction. The conformational change also permits formation of additional weak bonding interactions in the transition state. In either case, the new enzyme conformation has enhanced catalytic properties. As we have seen, induced fit is a common feature of the reversible binding of ligands to proteins (Chapter 5). Induced fit is also important in the interaction of almost every enzyme with its substrate. Specific Catalytic Groups Contribute to Catalysis In most enzymes, the binding energy used to form the ES complex is just one of several contributors to the overall catalytic mechanism. Once a substrate is bound to an enzyme, properly positioned catalytic functional groups aid in the cleavage and formation of bonds by a variety of mechanisms, including general acid-base catalysis, covalent catalysis, and metal ion catalysis. These are distinct from mechanisms based on binding energy, because they generally involve transient covalent interaction with a substrate or group transfer to or from a substrate. General Acid-Base Catalysis Many biochemical reactions involve the formation of unstable charged intermediates that tend to break down rapidly to their constituent reactant species, thus impeding the reaction (Fig. 6–8). Charged intermediates can often be stabilized by the transfer of protons to or from the substrate or intermediate to form a species that breaks down more readily to products. For nonenzymatic reactions, the proton transfers can involve either the constituents of water alone or other weak proton donors or acceptors. Catalysis of this type that uses only the H (H3O) or OH ions present in water is referred to as specific acid-base catalysis. If protons are transferred between the intermediate and water faster than the intermediate breaks down to reactants, the intermediate is effectively stabilized every time it forms. No additional catalysis mediated by other proton acceptors or donors will occur. In many cases, however, water is not enough. The term general acid-base catalysis refers to proton transfers mediated by other classes of molecules. For nonenzymatic reactions in aqueous solutions, this occurs only when the unstable reaction intermediate breaks down to reactants faster than protons can be transferred to or from water. Many weak organic acids can supplement water as proton donors in this situation, or weak organic bases can serve as proton acceptors. In the active site of an enzyme, a number of amino acid side chains can similarly act as proton donors and acceptors (Fig. 6–9). These groups can be precisely positioned in an enzyme active site to allow proton transfers, providing rate enhancements of the order of 102 to 105 . This type of catalysis occurs on the vast majority of enzymes. In fact, proton transfers are the most common biochemical reactions. Covalent Catalysis In covalent catalysis, a transient covalent bond is formed between the enzyme and the substrate. Consider the hydrolysis of a bond between groups A and B: H2O AOB On A B In the presence of a covalent catalyst (an enzyme with a nucleophilic group X:) the reaction becomes H2O AOB X On AOX B On A X B 200 Chapter 6 Enzymes O CH3 C CH3 CH3 OR O CH3 C O k (M1 s1 ) OR C C O O O Reaction Rate enhancement (a) 1 k (s1 ) OR (b) O C C C C 105 M OR O O O O O k (s1 ) OR (c) 108 M C O O C O OR O C O C O O O FIGURE 6–7 Rate enhancement by entropy reduction. Shown here are reactions of an ester with a carboxylate group to form an anhydride. The R group is the same in each case. (a) For this bimolecular reaction, the rate constant k is second order, with units of M1 s 1 . (b) When the two reacting groups are in a single molecule, the reaction is much faster. For this unimolecular reaction, k has units of s1 . Dividing the rate constant for (b) by the rate constant for (a) gives a rate enhancement of about 105 M. (The enhancement has units of molarity because we are comparing a unimolecular and a bimolecular reaction.) Put another way, if the reactant in (b) were present at a concentration of 1 M, the reacting groups would behave as though they were present at a concentration of 105 M. Note that the reactant in (b) has freedom of rotation about three bonds (shown with curved arrows), but this still represents a substantial reduction of entropy over (a). If the bonds that rotate in (b) are constrained as in (c), the entropy is reduced further and the reaction exhibits a rate enhancement of 108 M relative to (a). 8885d_c06_190-237 1/27/04 7:13 AM Page 200 mac76 mac76:385_reb: