title: Crystal structure of rat trypsin-S195C at -150 degrees C. Analysis of low activity of recombinant and semisynthetic thiol proteases.
abstract: The X-ray crystal structure of trypsin-S195C, a rat anionic trypsin mutant in which the active site serine has been replaced by cysteine, was determined at -150 degrees C and room temperature to 1.6 A resolution, R = 15.4% and 1.8 A resolution, R = 15.0%, respectively. Cryo-crystallography was employed to improve the quality of the diffraction data and the resulting structure by eliminating radiation damage and decreasing atomic thermal motion. The average temperature factor decreased by 10 A2 relative to that of the room temperature structure. No radiation-induced decay of the data was detected. The side-chains of the catalytic cysteine and histidine of trypsin-S195C are found with 25% occupancy in secondary orientations rotated 104 degrees and 90 degrees out of the active site, respectively. These alterations, as well as more subtle changes in the active site may be caused by the oxidation of the catalytic sulfur to sulfenic acid. The position of the carbonyl carbon of the tetrahedral intermediate analog, p-amidinophenylpyruvic acid, modeled into trypsin-S195C, is 1.1 A from the catalytic sulfur. The large size and altered approach of the catalytic sulfur to substrates could account for the observed low catalytic activity relative to wild-type trypsin. In addition to the benzamidine in the specificity pocket, two additional binding sites for benzamidine are characterized. One of these mediates an intermolecular contact that appears to maintain the crystal lattice.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
authors.forename: M E
authors.initials: ME
authors.lastname: Wilke
authors.forename: J N
authors.initials: JN
authors.lastname: Higaki
authors.forename: C S
authors.initials: CS
authors.lastname: Craik
authors.forename: R J
authors.initials: RJ
authors.lastname: Fletterick
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003545:Cysteine; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D005615:Freezing; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics; D014961:X-Ray Diffraction / Q000379:methods
chemical_list: D011994:Recombinant Proteins; D012694:Serine; D014357:Trypsin; D003546:Cysteine Endopeptidases; D003545:Cysteine