Proton donors, carboxypeptidase

The collapse of the proteolytic tetrahedral intermediate of the promoted-water pathway requires a proton donor in order to facilitate the departure of the leaving amino group. Rees and Lipscomb (1982) considered Glu-270, but favored Tyr-248 for this role, but Monzingo and Matthews (1984) fully elaborated on a role for Glu-270 of carboxypeptidase A and Glu-143 of thermolysin as intermediate proton donors. This proposal for carboxypeptidase A is corroborated by the near-normal activity observed for the Tyr-248- Phe mutant of rat carboxypeptidase A (Garden et al, 1985 Hilvert et al, 1986) and is reflected in the mechanistic scheme of Fig. 31 (Christianson and Lipscomb, 1989). Mock (1975) considered Glu-270 a proton donor in the carboxypeptidase A mechanism, but his mechanism does not favor a Glu-270/zinc-promoted water molecule as the hydrolytic nucleophile. Schepartz and Breslow (1987) observed that Glu-270 may mediate an additional proton transfer in the generation of the Pi product carboxylate. 

On the basis of chemical modification studies, Tyr 198 of carboxypeptidase A was proposed to act as a proton donor (i.e., a general acid) in the mechanism of catalysis. However, when Tyr 198 was replaced with Phe by means of site-directed mutagenesis, the modified enzyme retained substantial enzymatic activity, indicating that the tyrosyl hydroxyl may not have a specific role in catalysis. 

The addition or removal of a proton can promote electron flow, i.e., bond formation and breakage, during the reaction. As far as enzymes are concerned, only general acid or base catalysis can occur, because enzymes have no means of concentrating or OH ions. A number of amino acid side chains can act as proton donors or acceptors, and in many Zn-dependent enzymes, such as carboxypeptidase. the metal ion acts as an effective Lewis acid to enhance polarization of the carbonyl moiety of the amide bond. Among the amino acids His (which has a pKa generally close to 7) plays an especially important role in enzyme catalysis, because at neutral pH, there is a good balance between its protonated and deprotonated forais. In most cases, enzymes have suitably positioned pairs of side chains to provide push and pull of electrons. 

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