MO-STUDIES OF ENZYME REACTION-MECHANISMS .1. MODEL MOLECULAR-ORBITAL STUDY OF THE CLEAVAGE OF PEPTIDES BY CARBOXYPEPTIDASE-A

Ab initio and semiempirical (AM1) molecular orbital theory has been used to model the cleavage of formamide at the active site of carboxypeptidase A. The model active site consists of a zinc dication coordinated to two imidazoles, an acetate, a water with a hydrogen-bonded formate, and a formamide molecule as model substrate. AM1 has been compared with ab initio theory for the coordination of water and formamide to Zn++ and found to give excellent energetic results. The course of the amide cleavage was therefore calculated with AM1. The first step of the reaction is the dissociation of the zine-coordinated water to give an active ZnOH+ species. The remote formate acts as proton acceptor. This process has an activation energy of only 4.6 kcal mol-1. The next and rate-determining step is the concerted addition of the ZnOH+ moiety to the formamide C=O bond. The Zn-0 distance in the transition state is more than 3 angstrom. In four further steps, the amide nitrogen is protonated and the C-N bond cleaved. The net activation energy for the entire process is 15.5 kcal mol-1 relative to the active site model and 19.6 kcal mol-1 relative to the most stable point on the calculated reaction profile.