213 figure 231 stereo view of the active site of

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""" 213 Figure 2.31 Stereo view of the active site of thennolysin. 139 B. Ester Hydrolysis and Phosphoryl Transfer Hydrolysis of carboxylic and phosphoric esters is also a slow process at neutral pH, and is catalyzed by acids and bases by mechanisms similar to those in- volved in amide and peptide hydrolysis. Metal ions are also good catalysts of both carboxylic and phosphoric ester hydrolysis, typically with rate increases much higher than those observed for hydrolysis of amides or peptides (Table
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86 2 I THE REACTION PATHWAYS OF ZINC ENZYMES AND RELATED BIOLOGICAL CATALYSTS 2.8). The ability of metal ions to coordinate to the carbonyl oxygen-which is higher in amides than in esters-is inversely correlated with their catalytic prop- erties, perhaps because the main role of the metal ion is not in polarizing the carbonyl group, but in providing a metal-coordinated hydroxide as the attacking nucleophile. 108 For the hydrolysis of phosphate esters, it is difficult to draw conclusions based on experience with carboxylic esters, because, although the coordinating ability of the phosphoric oxygen may be higher, thus favoring the polarizing role of the metal, the nucleophilic attack is also likely to be easier, because the energy of the trigonal bipyramidal intermediate is probably rather low. Base-catalyzed hydrolysis of phosphate esters occurs with inversion of con- figuration, and this supports the existence of a trigonal bipyramidal intermedi- ate. 140 The metal acts both as activator of substrate through binding and as Lewis acid to provide the OH moiety for the nucleophilic attack: o OH OH \ I / H-OI - O...-P-OR ~ O-P-OR ~ o-P--.. o + RO- (2.20) / / / /\ / \ M----O M-O 0 M 0 As with peptide hydrolysis, several enzyme systems exist that catalyze car- boxylic and phosphoric ester hydrolysis without the need for a metal ion. They generally involve a serine residue as the nuc1eophile; in turn, serine may be activated by hydrogen-bond formation-or even proton abstraction-by other acid-base groups in the active site. The reaction proceeds to form an acyl- or phosphoryl-enzyme intermediate, which is then hydrolyzed with readdition of a proton to the serine oxygen. Mechanisms of this type have been proposed for chymotrypsin. 141 In glucose-6-phosphatase the nuc1eophile has been proposed to be a histidine residue. 142 Again by analogy with peptide hydrolysis, metalloenzymes catalyzing ester hydrolysis may take advantage of additional chemical features provided by amino- acid residues present in the active-site cavity. This situation occurs with car- boxypeptidase,143 which shows esterase activity in vitro. Although the rate-lim- iting steps for carboxylic esters and peptides may differ, several features, such as the pH dependences of k cat and K m and the presence of two spectroscopically observable intermediates, point to substantially similar mechanisms. On the other hand, carboxylic ester hydrolysis catalyzed by carbonic anhydrase seems to rely on fewer additional features of the active-site cavity, perhaps only on the pres- ence of
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