The role of disulfide bond C191-C220 in trypsin and chymotrypsin

Biochem Biophys Res Commun. 1997 Jan 23;230(3):592-6. doi: 10.1006/bbrc.1996.6009.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Catalysis
  • Cattle
  • Chymotrypsin / chemistry*
  • Chymotrypsin / genetics
  • Conserved Sequence
  • Cysteine / chemistry*
  • Cysteine / genetics
  • Disulfides*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Rats
  • Substrate Specificity
  • Trypsin / chemistry*
  • Trypsin / genetics

Substances

  • Disulfides
  • Chymotrypsin
  • Trypsin
  • Cysteine