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)
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: | https://repository.cshl.edu/id/eprint/32341 |
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