1GBT

STRUCTURE OF AN ACYL-ENZYME INTERMEDIATE DURING CATALYSIS: (GUANIDINOBENZOYL) TRYPSIN

Summary for 1GBT

• Entry DOI: 10.2210/pdb1gbt/pdb
• Descriptor: BETA-TRYPSIN, CALCIUM ION, SULFATE ION, ... (5 entities in total)
• Functional Keywords: hydrolase(serine proteinase)
• Biological source: Bos taurus (cattle)
• Cellular location: Secreted, extracellular space: P00760
• Total number of polymer chains: 1
• Total formula weight: 23735.67

Authors

Singer, P.T.,Sweet, R.M. (deposition date: 1991-09-17, release date: 1994-01-31, Last modification date: 2024-11-06)

Primary citation

Mangel, W.F.,Singer, P.T.,Cyr, D.M.,Umland, T.C.,Toledo, D.L.,Stroud, R.M.,Pflugrath, J.W.,Sweet, R.M.
Structure of an acyl-enzyme intermediate during catalysis: (guanidinobenzoyl)trypsin.
Biochemistry, 29:8351-8357, 1990

PubMed 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)

PubMed: 2252895
DOI: 10.1021/bi00488a022

Experimental method

X-RAY DIFFRACTION (2 Å)