The crystal structure of the bovine zinc metalloproteinase carboxypeptidase A (CPA) has been refined to 1.25 A resolution based on room-temperature X-ray synchrotron data. The significantly improved structure of CPA at this resolution (anisotropic temperature factors, R factor = 10.4%, R(free) = 14.5%) allowed the modelling of conformational disorders of side chains, improved the description of the protein solvent network (375 water molecules) and provided a more accurate picture of the interactions between the active-site zinc and its ligands. The calculation of standard uncertainties in individual atom positions of the refined model of CPA allowed the deduction of the protonation state of some key residues in the active site and confirmed that Glu72 and Glu270 are negatively charged in the resting state of the enzyme at pH 7.5. These results were further validated by theoretical calculations that showed significant reduction of the pK(a) of these side chains relative to solution values. The distance between the zinc-bound solvent molecule and the metal ion is strongly suggestive of a neutral water molecule and not a hydroxide ion in the resting state of the enzyme. These findings could support both the general acid/general base mechanism, as well as the anhydride mechanism suggested for CPA.

ABSTRACT: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-(CH 2 CO)-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.

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.

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