Mechanism of action of zinc proteinases: A MNDO/d/H study of alternative general‐acid general‐base catalytic pathways for carboxypeptidase‐A

Two alternative pathways for peptide cleavage by water, both of the general‐acid general‐base type, have been followed by semiempirical theoretical calculations on a model of the active site. The system of 120 atoms has been carved out of high resolution X‐ray coordinates of a carboxypeptidase A (CPA) complex with a ketomethylene inhibitor, pyroglutamic‐N‐Phe‐(CH2CO)‐Phe‐OH. The method employed was a combination of MNDO/d and MNDO/H which, together, enable one to deal with the effect of zinc and of multiple hydrogen bond interactions, respectively. The first step in both pathways is nucleophilic attack by a hydroxide on the peptide carbonyl, and the second is proton transfer to the nitrogen of the peptide. This second step presents the highest energy barrier for the reaction. Peptide bond cleavage is spontaneous subsequent to proton transfer. The two alternative paths differ little in barrier heights, but the thermodynamic enthalpy difference for the path of one mechanism is some 20 kcal/mol more stable than for the other. The first mechanism is the one proposed by Lipscomb (Acc Chem Res 1989, 22, 62–69) and the second, less stabilizing mechanism was proposed by Mock (J Biol Chem 1991, 266, 6369–6400). Under kinetic control, both reactions are feasible, and new experiments should be designed in order to clarify if only one of the two is operating under most of the relevant conditions.