Ab initio and semiempirical (AMl) 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 zinc-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-'. 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 A .
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-' relative to the active site model and 19.6 kcal mol-' relative to the most stable point on the calculated reaction profile.
'Author to whom all correspondence should be addressed. and Lipscomb6 reported an MO study on the hydrolysis of peptides by CPA. Osman and Weinstein7 examined models for active sites of metalloenzymes and compared zinc-and beryllium-containing complexes by LCAO-SCF calculations, and Christianson and Alexander8 studied the role played by carboxylate-histidine-zinc interactions in protein structures and function. Pardo et al.9 theoretically examined models for proton transfer in biological systems. However, none of these publications proposed a full reaction path for the cleavage of peptides by CPA in an active-site model. This article reports a combined ab initio and AM1 study of the mechanism of peptide cleavage by CPA. We used an active-site model similar to that used by Merz et al. and find significant parallels to their calculated mode of activity for carbonic anhydrase .
Of the many zinc peptidases, thermolysin (Tln) and CPA are the best characterized. In both cases, X-ray structures of enzymehnhibitor complexes are available.
These structures formed the basis for our active site model. CPA catalyzes the hydrolysis of C-terminal amino acids and shows a strong preference for cleaving C-terminal residues that contain aromatic or branched chains. l2 Three realistic mechanisms have been proposed to explain the action of CPA: the anhydride mechanism,