title: The role of disulfide bond C191-C220 in trypsin and chymotrypsin.
abstract: Serine proteases of the chymotrypsin family contain three conserved disulfide bonds: C42-C58, C168-C182, and C191-C220. C191-C220 connects the loops around the substrate binding pocket. Using site directed mutagenesis, cysteines of this disulfide bridge were replaced by alanines in trypsin, in chymotrypsin, and in Tr-->Ch-[S1+L1+L2+Y172W], a mutant trypsin with high chymotrypsin like activity. The functional role of this "active site" disulfide was assessed by comparing the catalytic properties of wild-type and mutant enzymes. Its removal from all three proteases caused a decrease in kcat/KM of two to three orders of magnitude, mainly as a consequence of a dramatic increase in KM. The pH dependence of the activity also changed: the rather wide pH optimum, characteristic of the wild-type enzymes (especially trypsin), narrowed since the pKa in the alkaline region shifted downwards. Results show that C191-C220 is necessary for the high activity of both trypsin and chymotrypsin. By contrast, elimination of this disulfide bridge greatly decreased the specificity of trypsin and of Tr-->Ch-[S1+L1+L2+Y172W], but had no significant change on that of chymotrypsin.
authors.affiliation: Department of Biochemistry, Eötvös University, Budapest, Hungary.
authors.forename: E
authors.initials: E
authors.lastname: Várallyay
authors.forename: Z
authors.initials: Z
authors.lastname: Lengyel
authors.forename: L
authors.initials: L
authors.lastname: Gráf
authors.forename: L
authors.initials: L
authors.lastname: Szilágyi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry* / Q000235:genetics; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics
chemical_list: D004220:Disulfides; D002918:Chymotrypsin; D014357:Trypsin; D003545:Cysteine