Ala226, to Gly and Ser189 to Asp mutations convert rat chymotrypsin B to a trypsin-like protease

Balázs Jelinek, József Antal, István Venekei, László Gráf

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

In a previous successful attempt to convert trypsin to a chymotrypsin-like protease, 15 residues of trypsin were replaced with the corresponding ones in chymotrypsin. This suggests a complex mechanism of substrate recognition instead of a relatively simple one that only involves three sites, residues 189, 216 and 226. However, both trypsin→elastase and chymotrypsin→trypsin conversion experiments carried out according to the complex model resulted in non-specific proteases with low catalytic activity. Chymotrypsin used in the latter studies was of B-type, containing an Ala residue at position 226. Trypsins, however, contain a conserved Gly at this site. The substantially decreased trypsin-like activity of the G226A trypsin mutant also suggests a specific role for this site in substrate binding. Here we investigate the role of site 226 by introducing the A226G substitution into chymotrypsin→ trypsin mutants which were constructed according to both the simple (S189D mutant) and the complex model (S1 mutant) of specificity determination. The kinetic parameters show that the A226G substitution in the S1 mutant increased the chymotrypsin-like activity, while the trypsin-like activity did not change. In contrast, this substitution in the S189D chymotrypsin mutant resulted in a 100-fold increase in trypsin-like activity and a trypsin-like specificity profile as tested on a competing oligopeptide substrate library. Additionally, the S189D+A226G mutant is the first trypsin-like chymotrypsin that undergoes autoactivation, an exclusive property of trypsinogen among pancreatic serine proteases.

Original languageEnglish
Pages (from-to)127-131
Number of pages5
JournalProtein Engineering, Design and Selection
Volume17
Issue number2
DOIs
Publication statusPublished - Feb 2004

Fingerprint

Trypsin
Rats
Peptide Hydrolases
Substitution reactions
Mutation
Chymotrypsin
Substrates
Kinetic parameters
Catalyst activity
Chymases
Trypsinogen
Oligopeptides
chymotrypsin B
Serine Proteases
Experiments
Libraries

Keywords

  • Autoactivation
  • Serine protease
  • Substrate specificity

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology

Cite this

Ala226, to Gly and Ser189 to Asp mutations convert rat chymotrypsin B to a trypsin-like protease. / Jelinek, Balázs; Antal, József; Venekei, István; Gráf, László.

In: Protein Engineering, Design and Selection, Vol. 17, No. 2, 02.2004, p. 127-131.

Research output: Contribution to journalArticle

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