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The detection, accumulation and stabilization of a tetrahedral intermediate in trypsin catalysis

Authors
Journal
Biochemical and Biophysical Research Communications
0006-291X
Publisher
Elsevier
Publication Date
Volume
93
Issue
2
Identifiers
DOI: 10.1016/0006-291x(80)91095-5

Abstract

Abstract The reaction of trypsin with N α-carbobenzoxy-L-lysine p-nitroanilide has been studied in the 0 to −60°C temperature region, over a wide range of pH ★ values, using aqueous-dimethyl sulfoxide cryosolvent. Spectrophotometric and kinetic evidence for the detection and stabilization of a tetrahedral intermediate is presented. The rate of formation of this intermediate, and the concentration accumulated, were pH ★-dependent. At pH ★ ≤ 6 (< −40°C) essentially no tetrahedral adduct is formed or accumulated. Instead another intermediate, resembling the substrate in its spectral properties, is detected.

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