Trypsin specificity increased through substrate-assisted catalysis

D. R. Corey, W. S. Willett, G. S. Coombs, C. S. Craik

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

Histidine 57 of the catalytic triad of trypsin was replaced with alanine to determine whether the resulting variant would be capable of substrate-assisted catalysis [Carter, P., & Wells, J. A. (1987) Science 237, 394-9]. A 2.5-fold increase in kcat/Km was observed on tri- or tetrapeptide substrates containing p-nitroanilide leaving groups and histidine at P2. In contrast, hydrolysis of peptide substrates extending from P6 to P6′ is improved 70-300-fold by histidine in the P2 or P1′ position. This preference creates new protease specificities for sequences HR↓, R↓H, HK↓, and K↓H. The ability of histidine from either the P2 or the P1′ position of substrate to participate in catalysis emphasizes the considerable variability of proteolytically active orientations which can be assumed by the catalytic triad. Trypsin H57A is able to hydrolyze fully folded ornithine decarboxylase with complete specificity at a site containing the sequence HRH. Trypsin H57A was compared to enteropeptidase in its ability to cleave a propeptide from trypsinogen. Trypsin H57A cleaved the propeptide of a variant trypsinogen containing an introduced FPVDDDHR cleavage site only 100-fold slower than enteropeptidase cleaved trypsinogen. The selective cleavage of folded proteins suggests that trypsin H57A can be used for specific peptide and protein cleavage. The extension of substrate-assisted catalysis to the chymotrypsin family of proteolytic enzymes indicates that it may be possible to apply this strategy to a wide range of serine proteases and thereby develop various unique specificities for peptide and protein hydrolysis.

Original languageEnglish (US)
Pages (from-to)11521-11527
Number of pages7
JournalBiochemistry
Volume34
Issue number36
StatePublished - 1995

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Catalysis
Trypsin
Trypsinogen
Histidine
Enteropeptidase
Substrates
Peptides
Hydrolysis
Peptide Hydrolases
Proteins
Ornithine Decarboxylase
Chymotrypsin
Serine Proteases
Alanine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Corey, D. R., Willett, W. S., Coombs, G. S., & Craik, C. S. (1995). Trypsin specificity increased through substrate-assisted catalysis. Biochemistry, 34(36), 11521-11527.

Trypsin specificity increased through substrate-assisted catalysis. / Corey, D. R.; Willett, W. S.; Coombs, G. S.; Craik, C. S.

In: Biochemistry, Vol. 34, No. 36, 1995, p. 11521-11527.

Research output: Contribution to journalArticle

Corey, DR, Willett, WS, Coombs, GS & Craik, CS 1995, 'Trypsin specificity increased through substrate-assisted catalysis', Biochemistry, vol. 34, no. 36, pp. 11521-11527.
Corey DR, Willett WS, Coombs GS, Craik CS. Trypsin specificity increased through substrate-assisted catalysis. Biochemistry. 1995;34(36):11521-11527.
Corey, D. R. ; Willett, W. S. ; Coombs, G. S. ; Craik, C. S. / Trypsin specificity increased through substrate-assisted catalysis. In: Biochemistry. 1995 ; Vol. 34, No. 36. pp. 11521-11527.
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