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

E. Várallyay, Zsolt Lengyel, L. Gráf, László Szilágyi

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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 sub strate 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 K(cat)/K(M) of two to three orders of magnitude, mainly as a consequence of a dramatic increase in K(M). The pH dependence of the activity also changed: the rather wide pH optimum, characteristic of the wild-type enzymes (especially trypsin), narrowed since the pK(a) 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.

Original languageEnglish
Pages (from-to)592-596
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume230
Issue number3
DOIs
Publication statusPublished - Jan 23 1997

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Chymotrypsin
Disulfides
Trypsin
Mutagenesis
Serine Proteases
Enzymes
Site-Directed Mutagenesis
Alanine
Cysteine
Catalytic Domain
Cats
Peptide Hydrolases
trypsin drug combination chymotrypsin

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

The role of disulfide bond C191-C220 in trypsin and chymotrypsin. / Várallyay, E.; Lengyel, Zsolt; Gráf, L.; Szilágyi, László.

In: Biochemical and Biophysical Research Communications, Vol. 230, No. 3, 23.01.1997, p. 592-596.

Research output: Contribution to journalArticle

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