Theoretical Studies of Catalysis by Carboxypeptidase A: Could Gas-Phase Calculations Support a Mechanism?

Abstract

We compare recent quantum mechanical computations of alternative reaction pathways for carboxypeptidase A, a zinc proteinase, in an "enzyme environment" to similar calculations in the "gas phase" that include the minimal chemical entities that are required for a non-catalytic reaction. The main question that we address is whether anything may be learned from such reduced representations. Two general acid-general base alternative pathways and one nucleophilic pathway are compared. The original calculations were run on a relatively large model (120 atoms) of the active site of carboxypeptidase A which included zinc and its ligands, as well as the residues Arg145, Arg127, Glu270, a water molecule and a model dipeptide. The "gas-phase" pathways include only the dipeptide, water and Glu270 and serve as models for the non-catalytic pathway. The calculations were performed by semiempirical MNDO/H/d that includes modifications for d-orbital representations as well as for intra- and intermolecular multiple H-bond formation. The gas-phase results strengthen our previous conclusion about the preference for general acid-general base pathways for peptide cleavage by carboxypeptidase A rather than a "direct nucleophilic" pathway. The bottleneck of the reaction is proton transfer to the nitrogen in the peptide bond, preceding the peptide cleavage.

On the origin of the catalytic power of carboxypeptidase A and other metalloenzymes

“…Goldblum and co-workers performed a semi-empirical QM study on the alternative mechanisms for zinc proteinases 58–60. Their results suggest that the anhydride pathway is not a viable candidate to be the mechanism of cleavage of peptides by CPA 60 and that both GAGB mechanisms are feasible 58,59. Banci et al 61 demonstrated the mobility of Arg127 and Tyr248 and suggested 62 that both “nucleophilic” and “water-promoted” pathways are structurally feasible, although the water-promoted mechanism is favored energetically.…”