An investigation into the minimum requirements for peptide hydrolysis by mutation of the catalytic triad of trypsin

David R. Corey, Charles S. Craik

Research output: Contribution to journalArticle

132 Citations (Scopus)

Abstract

The catalytic triad of rat anionic trypsin has been systematically altered by site-directed mutagenesis to determine the activity of alternate combinations of amino acids toward the hydrolysis of peptide bonds. Genetically modified rat trypsins H57A, H57D, H57E, H57K, H57R, H57A/D102N, H57D/D102N, H57L/D102N, H57K/D102N, D102N, S195A, S195T, and H57A/D102N/S195A have been generated. Rigorous steps were taken to show that the resultant catalysis was due to the mutant enzymes and not contaminants. Each of the variants exhibit measurable activity toward the activated amide substrate Z-GPR-AMC. At pH 8.0 kcat ranges from 0.011 to 1.3 min-1 (0.0004-0.04% of wild-type). At pH 10.5 kcat ranges from 0.012 to 140 m-1 (0.0004-5% of wild-type). The mutant trypsins were subsequently assayed for their ability to hydrolyze the unactivated amide linkages of protein substrates. Trypsins D102N, H57K, and H57K/D102N exhibited the highest level of activity. The kcat for the D102N enzyme was 4 h-1 (0.003% of wild-type). The H57A/D102N double mutant was not as active but was chosen for further study since it was the simplest trypsin to exhibit peptidase activity. Its kcat was ∼ 0.1-0.2 h-1 at pH 8.0 and 0.7 h-1 at pH 10.1. These experiments demonstrate that an intact catalytic triad is not a requirement for peptide bond cleavage and that designed serine peptidases need not include a catalytic histidine or aspartic acid.

Original languageEnglish (US)
Pages (from-to)1784-1790
Number of pages7
JournalJournal of the American Chemical Society
Volume114
Issue number5
StatePublished - 1992

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Amides
Trypsin
Peptides
Rats
Hydrolysis
Enzymes
Ground penetrating radar systems
Mutagenesis
Mutation
Substrates
Catalysis
Amino acids
Impurities
Proteins
Peptide Hydrolases
Acids
Site-Directed Mutagenesis
Histidine
Aspartic Acid
Experiments

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

An investigation into the minimum requirements for peptide hydrolysis by mutation of the catalytic triad of trypsin. / Corey, David R.; Craik, Charles S.

In: Journal of the American Chemical Society, Vol. 114, No. 5, 1992, p. 1784-1790.

Research output: Contribution to journalArticle

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