CRYSTAL-STRUCTURE OF THE COMPLEX OF CARBOXYPEPTIDASE-A WITH A STRONGLY BOUND PHOSPHONATE IN A NEW CRYSTALLINE FORM - COMPARISON WITH STRUCTURES OF OTHER COMPLEXES

O-[[(lR)-[[N-(Phenylmethoxycarbonyl)-L-alanyl]amino]ethyl]hydroxyphosphinyl]-L-3-phenyllacetate [ZAAp(O)F], an analogue of (benzyloxycarbonyl)-Ala-Ala-Phe or (benzyloxycarbonyl)-Ala-Ala-phenyllactate, binds to carboxypeptidase A with great affinity (Ki = 3 pM). Similar phosphonates have been shown to be transition-state analogues of the CPA-catalyzed hydrolysis [Hanson, J. E., Kaplan, A. P., & Bartlett, P. A. (1989) Biochemistry 28, 6294-6305]. In the present study, the structure of the complex of this phosphonate with carboxypeptidase A has been determined by X-ray crystallography to a resolution of 2.0 Å. The complex crystallizes in the space group P212121 with cell dimensions a = 61.9 Å, b = 67.2 Å, and c = 76.2 A. The structure of the complex was solved by molecular replacement. Refinement of the structure against 20776 unique reflections between 10.0 and 2.0 Å yields a crystallographic residual of 0.193, including 140 water molecules. The two phosphinyl oxygens of the inhibitor bind to the active-site zinc at 2.2 Å on the electrophilic (Arg-127) side and 3.1 Å on the nucleophilic (Glu-270) side. Various features of the binding mode of this phosphonate inhibitor are consistent with the hypothesis that carboxypeptidase A catalyzed hydrolysis proceeds through a general-base mechanism in which the carbonyl carbon of the substrate is attacked by Zn-hydroxyl (or Zn-water). An unexpected feature of the bound inhibitor, the cis carbamoyl ester bond at the benzyloxycarbonyl linkage to alanine, allows the benzyloxycarbonyl phenyl ring of the inhibitor to interact favorably with Tyr-198. This complex structure is compared with previous structures of carboxypeptidase A, including the complexes with the potato inhibitor, a hydrated keto methylene substrate analogue, and a phosphonamidate inhibitor. Comparisons are also made with the complexes of thermolysin with some phosphonamidate inhibitors. © 1990, American Chemical Society.