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Title Structure of an acyl-enzyme intermediate during catalysis: (Guanidinobenzoyl)trypsin
Creator/Author Mangel, W.F. ; Singer, P.T. ; Cyr, D.M. ; Umland, T.C. ; Toledo, D.L. (Brookhaven National Lab., Upton, NY (USA)) ; Stroud, R.M. (Univ. of California, San Francisco (USA)) ; Pflugrath, J.W. ; Sweet, R.M. (Cold Spring Harbor Lab., NY (USA))
Publication Date1990 Sep 11
OSTI IdentifierOSTI ID: 5761306
Other Number(s)ISSN0006-2960; CODEN: BICHA
Resource TypeJournal Article
Resource RelationBiochemistry ; Vol/Issue: 29:36; DOE Project
Subject550602 -- Medicine-- External Radiation in Diagnostics-- (1980-); TRYPSIN-- CATALYSIS;TRYPSIN-- X-RAY DIFFRACTION; BIOCHEMISTRY;ENZYME ACTIVITY;HYDROLYSIS;MOLECULAR STRUCTURE;X-RAY DIFFRACTOMETERS
Related SubjectCHEMICAL REACTIONS;CHEMISTRY;COHERENT SCATTERING;DECOMPOSITION;DIFFRACTION;DIFFRACTOMETERS;ENZYMES;HYDROLASES;LYSIS;MEASURING INSTRUMENTS;PEPTIDE HYDROLASES;SCATTERING;SERINE PROTEINASES;SOLVOLYSIS
Description/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.^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 {angstrom}.^The refined model of (guanidinobenzoyl)trypsin provides insight into the structural basis for its slow rate of deacylation, which in solution at 25{degree}C and pH 7.4 exhibits a t{sub 1/2} of 12h.^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.^Coupled with an apparent 35% decrease in the overall temperature factor of the acyl-enzyme relative to the native structure, the tight packing and rigidity of all atoms in the active site, including solvent, prevent disordered water molecules from easily approaching the carbonyl carbon atom via diffusion.
Country of PublicationUnited States
LanguageEnglish
FormatPages: 8351-8357
System Entry Date2001 May 13

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