title: Structural and functional integrity of specificity and catalytic sites of trypsin.
abstract: The aspartic acid residue at the bottom of the substrate-binding pocket of trypsin was replaced by glutamic acid through site-directed mutagenesis. The wild-type (Asp-189) and mutant (Glu-189) trypsinogens were expressed in E. coli, purified to homogeneity, activated by enterokinase, and tested on a series of fluorogenic tetrapeptide substrates. The substrates were of the general formula succinyl-Ala-Ala-Pro-X-AMC, where AMC is 7-amino-4-methylcoumarin and X is Lys, Arg, or Orn (ornithine). As compared to Asp-189 trypsin, the activity of Glu-189 trypsin on lysyl and arginyl substrates decreased by 3-4 orders of magnitude while its Km values did not significantly change. Lengthening the side-chain of Asp-189 by one methylene group could not be compensated for by shortening the side-chain of the substrate, since Glu-189 trypsin had no measurable activity on the ornithyl substrate. The replacement of Asp-189 with glutamic acid at the base of the substrate-binding pocket of trypsin appears to distort the structure of the critical transition-state complex. This could happen by disrupting interactions normally associated with Asp-189, and by altering the relative position of the scissile peptide bond in the active site of the enzyme.
authors.affiliation: L. Eötvös University, Biochemistry Department, Budapest, Hungary.
authors.forename: L
authors.initials: L
authors.lastname: Gráf
authors.forename: G
authors.initials: G
authors.lastname: Hegyi
authors.forename: I
authors.initials: I
authors.lastname: Likó
authors.forename: J
authors.initials: J
authors.lastname: Hepp
authors.forename: K
authors.initials: K
authors.lastname: Medzihradszky
authors.forename: C S
authors.initials: CS
authors.lastname: Craik
authors.forename: W J
authors.initials: WJ
authors.lastname: Rutter
mesh_terms: D001224:Aspartic Acid; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008962:Models, Theoretical; D012694:Serine; D014357:Trypsin / Q000235:genetics / Q000378:metabolism*; D014362:Trypsinogen / Q000235:genetics / Q000378:metabolism
chemical_list: D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; D014362:Trypsinogen; D014357:Trypsin