6CPA

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

Summary for 6CPA

• Entry DOI: 10.2210/pdb6cpa/pdb
• Descriptor: CARBOXYPEPTIDASE A, ZINC ION, O-(((1R)-((N-PHENYLMETHOXYCARBONYL-L-ALANYL)AMINO)ETHYL)HYDROXYPHOSPHONO)-L-BENZYLACETIC ACID, ... (4 entities in total)
• Functional Keywords: hydrolase (c-terminal peptidase)
• Biological source: Bos taurus (cattle)
• Cellular location: Secreted, extracellular space: P00730
• Total number of polymer chains: 1
• Total formula weight: 34986.30

Authors

Kim, H.,Lipscomb, W.N. (deposition date: 1990-02-15, release date: 1991-10-15, Last modification date: 2024-11-06)

Primary citation

Kim, H.,Lipscomb, W.N.
Crystal structure of the complex of carboxypeptidase A with a strongly bound phosphonate in a new crystalline form: comparison with structures of other complexes.
Biochemistry, 29:5546-5555, 1990

PubMed Abstract: O-[[(1R)-[[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 A. The complex crystallizes in the space group P2(1)2(1)2(1) with cell dimensions a = 61.9 A, b = 67.2 A, and c = 76.2 A. The structure of the complex was solved by molecular replacement. Refinement of the structure against 20,776 unique reflections between 10.0 and 2.0 A 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 A on the electrophilic (Arg-127) side and 3.1 A 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.

PubMed: 2386784
DOI: 10.1021/bi00475a019

Experimental method

X-RAY DIFFRACTION (2 Å)