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

Journal Article · Tue Sep 11 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00488a022· OSTI ID:5761306

Mangel, W F; Singer, P T; Cyr, D M; Umland, T C; Toledo, D L ^[1]; Stroud, R M ^[2]; Pflugrath, J W; Sweet, R M ^[3]

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1. Brookhaven National Lab., Upton, NY (USA)
2. Univ. of California, San Francisco (USA)
3. Cold Spring Harbor Lab., NY (USA)

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.

• OSTI ID: 5761306
• Journal Information: Biochemistry; (USA), Vol. 29:36; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

62 RADIOLOGY AND NUCLEAR MEDICINE
TRYPSIN
CATALYSIS
X-RAY DIFFRACTION
BIOCHEMISTRY
ENZYME ACTIVITY
HYDROLYSIS
MOLECULAR STRUCTURE
X-RAY DIFFRACTOMETERS
CHEMICAL REACTIONS
CHEMISTRY
COHERENT SCATTERING
DECOMPOSITION
DIFFRACTION
DIFFRACTOMETERS
ENZYMES
HYDROLASES
LYSIS
MEASURING INSTRUMENTS
PEPTIDE HYDROLASES
SCATTERING
SERINE PROTEINASES
SOLVOLYSIS
550602* - Medicine- External Radiation in Diagnostics- (1980-)