Structure of an acyl-enzyme intermediate during catalysis: (guanidinobenzoyl)trypsin

Mangel, W. F., Singer, P. T., Cyr, D. M., Umland, T. C., Toledo, D. L., Stroud, R. M., Pflugrath, J. W., Sweet, R. M. (September 1990) Structure of an acyl-enzyme intermediate during catalysis: (guanidinobenzoyl)trypsin. Biochemistry, 29 (36). pp. 8351-7. ISSN 0006-2960 (Print)0006-2960 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/2252895
DOI: 10.1021/bi00488a022

Abstract

The crystal and molecular structure of trypsin at a transiently stable intermediate step during catalysis has been determined by X-ray diffraction methods. Bovine trypsin cleaved the substrate p-nitrophenyl p-guanidinobenzoate during crystallization under conditions in which the acyl-enzyme intermediate, (guanidinobenzoyl)trypsin, was stable. Orthorhombic crystals formed in space group P2(1)2(1)2(1), with a = 63.74, b = 63.54, and c = 68.93 A. This is a crystal form of bovine trypsin for which a molecular structure has not been reported. Diffraction data were measured with a FAST (Enraf Nonius) diffractometer. The structure was refined to a crystallographic residual of R = 0.16 for data in the resolution range 7.0-2.0 A. The refined model of (guanidinobenzoyl)trypsin provides insight into the structural basis for its slow rate of deacylation, which in solution at 25 degrees C and pH 7.4 exhibits a t1/2 of 12 h. In addition to the rotation of the Ser-195 hydroxyl away from His-157, C beta of Ser-195 moves 0.7 A toward Asp-189 at the bottom of the active site, with respect to the native structure. This allows formation of energetically favorable H bonds and an ion pair between the carboxylate of Asp-189 and the guanidino group of the substrate. This movement is dictated by the rigidity of the aromatic ring in guanidinobenzoate--model-building indicates that this should not occur when arginine, with its more flexible aliphatic backbone, forms the ester bond with Ser-195. As a consequence, highly ordered water molecules in the active site are no longer close enough to the scissile ester bond to serve as potential nucleophiles for hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Item Type: Paper
Uncontrolled Keywords: Animals Benzoates/*metabolism Binding Sites Calcium/metabolism Catalysis Cattle Models, Molecular Molecular Structure Protein Conformation Trypsin/*metabolism Water/metabolism X-Ray Diffraction
Subjects: Investigative techniques and equipment > X-Ray Diffraction
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes
CSHL Authors:
Communities: CSHL labs
Depositing User: Matt Covey
Date: 11 September 1990
Date Deposited: 09 Feb 2016 16:45
Last Modified: 09 Feb 2016 16:45
Related URLs:
URI: http://repository.cshl.edu/id/eprint/32341

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