Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.8 (FAST)
 758 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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)
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PMID:Structure of an acyl-enzyme intermediate during catalysis: (guanidinobenzoyl)trypsin. 225 95

A novel strategy of macromolecular structure analysis is described which combines the use of monochromatic scanning Laue (SCL) and white-beam Laue (WBL) diffraction techniques. It provides, when applied with an area detector with on-line capabilities, a means of interactively determining and optimizing experimental parameters; it further makes rapid data evaluation feasible, also with off-line detector systems. These new procedures have been applied to a protein structure, beta-trypsin, using a FAST area detector (Enraf-Nonius) and image plates (Fuji) on a double-focusing synchrotron beamline at DORIS. Structure factors, which were derived from FAST Laue data, were empirically scaled by comparing equivalent reflections in different wavelength bins. A 2Fo-Fc difference Fourier map, which was calculated at 1.8 A resolution using these structure-factor moduli together with phases from the known structural model, showed well defined electron density distribution (R = 22%). Image-plate exposures showed diffraction to 1.2 A resolution. The effect of crystal mosaicity on the maximum wavelength bandwidth for Laue exposures has been investigated. SCL techniques, which involve rapid scanning (with a crystal or multilayer monochromator or a tunable undulator) through a defined wavelength range, extend the applicability of Laue techniques to crystals with broadened mosaic spread.
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PMID:Combined use of monochromatic and Laue diffraction techniques for macromolecular structure determination. 259 67

Orthorhombic crystals of the complex formed between bovine alpha-chymotrypsin and a recombinant human mucous proteinase inhibitor (SLPI) were grown. Data to 2.3 A resolution were collected on the area-detector diffractometer FAST. The crystal structure of the complex was solved by Patterson search techniques using chymotrypsin as a search model. A cyclic procedure of modeling and crystallographic refinement enabled the determination of the SLPI structure. The current crystallographic R-value is 0.19. SLPI has a boomerang-like shape with both wings comprising two well separated domains of similar architecture. In each domain the polypeptide chain is arranged like a stretched spiral. Two internal strands form a regular beta-hairpin loop which is accompanied by two external strands linked by the proteinase binding segment. The polypeptide segment of each domain is interconnected by four disulfide bridges with a connectivity pattern hitherto unobserved. The reactive site loop of the second domain has elastase and chymotrypsin binding properties. It contains the scissile peptide bond between Leu72I and Met73I and has a similar conformation to that observed in other serine proteinase protein inhibitors. Eight residues of this loop, two of the adjacent hairpin loop, the C-terminal segment and Trp30I are in direct contact with the cognate enzyme. The binding loop of the first domain (probably with anti-trypsin activity) is disordered due to proteolytic cleavage occurring in the course of crystallization.
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PMID:The 2.5 A X-ray crystal structure of the acid-stable proteinase inhibitor from human mucous secretions analysed in its complex with bovine alpha-chymotrypsin. 336 16