Carboxypeptidase Charge

FIGURE 27 19 Proposed mechanism of hydrolysis of a peptide catalyzed by carboxypeptidase A The peptide is bound at the active site by an ionic bond between its C terminal ammo acid and the positively charged side chain of arginine 145 Coordination of Zn to oxygen makes the carbon of the carbonyl group more positive and increases the rate of nucleophilic attack by water... 

Similar reaction mechanisms, involving general base and metal ion catalysis, in conjunction with an OH nucleophilic attack, have been proposed for thermolysin (Ref. 12) and carboxypeptidase A (Refs. 12 and 13). Both these enzymes use Zn2+ as their catalytic metal and they also have additional positively charged active site residues (His 231 in thermolysin and... 

In both carboxypeptidase A and thermolysin the active site Zn2+ is chelated by two imidazole groups and a glutamate side chain (Fig. 12-16). In carboxypeptidase A, Arg 145, Tyr 248, and perhaps Arg 127 form hydrogen bonds to the substrate. A water molecule is also bound to the Zn2+ ion. The presence of the positively charged side chain of Arg 145 and of a hydro-phobic pocket accounts for the preference of the enzyme for C-terminal amino acids with bulky, nonpolar side chains. The Zn2+ in thermolysin is also bound to two imidazole groups and that in D-alanyl-D-alanyl carboxypeptidase to three. 

The most important chemical function of Zn2+ in enzymes is probably that of a Lewis acid providing a concentrated center of positive charge at a nucleophilic site on the substrate/ This role for Zn2+ is discussed for carboxypeptidases (Fig.12-16) and thermolysin, alkaline phosphatase (Fig. 12-23),h RNA polymerases, DNA polymerases, carbonic anhydrase (Fig. 13-1),1 class II aldolases (Fig. 13-7), some alcohol dehydrogenases (Fig. 15-5), and superoxide dismutases (Fig.16-22). Zinc ions in enzymes can often be replaced by Mn2+, Co2+, and other ions with substantial retention of catalytic activity/ ... 

One obvious role for metals in metalloenzymes is to function as electrophilic catalysts, stabilizing the negative charges that are formed. In carboxypeptidase... 

Generally speaking, the role of the enzyme consists of the selective and specific attraction of substrate and the highly efficient catalysis. Every enzyme has its own characteristic feature for example, the specificity in the binding and a charge-relay action in the catalysis in a-chymotrypsin, the contribution of the imidazole moiety as an electron donor to the electrophilicity of zinc ion in carboxypeptidase, the change in the spin state and the reactivity of the transition metal ion by the coordination of the imidazole in the hemochrome. These typical characteristic features are the result of the cooperative actions of the constituents. 

The impetus for the newer work was the observation that benzyl clavulanate (11c) is a time-dependent inactivator of HLE (ICjq = 5 //M) whereas clavulanic acid (11b) is inactive [211, 212]. This finding led to the hypothesis that, since HLE is an endopeptidase whereas the bacterial serine proteinases are carboxypeptidases, quench(ing) the negative charge that the -lactam antibiotics normally require might yield HLE inhibitors. For synthetic reasons the group at Merck decided to use 7-aminocephalospo-ranic acids (1 Id) for most of their initial SAR studies. In contrast to the result with clavulanic acid, conversion of the cephalosporin 2 -carboxyl group in (1 Id) to an ester (He) was insufificent to transform the compound to an HLE inhibitor. 

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