Steady-state and pre-steady-state kinetics of the trypsin-catalysed hydrolysis of α-CBZ-l-lysine-p-nitrophenyl ester

P. Ascenzi, E. Menegatti, F. Bortolotti, M. Guarneri, E. Antonini

Research output: Contribution to journalArticlepeer-review

Abstract

The catalytic properties of bovine trypsin (EC 3.4.21.4) have been investigated using a synthetic chromogenic substrate: α-CBZ-l-lysine-p-nitrophenyl ester (ZLNPE). The use of ZLNPE allows the determination of trypsin down to a concentration of 2 · 10-9 M. Steady-state and pre-steady-state data have been analyzed in the framework of the minimum three-step mechanism: E + S ⇌ k-1 k+1 E · S ⇌ k-2 k+2 E · P + P1 ⇌ k-3 k+3 E + P2. The pH-dependence of the kinetic parameters shows that at acid pH values ({reversed tilde equals}2.6) the k+3 step is rate limiting in catalysis, whereas for pH values higher than 4.8 k+2 becomes rate limiting. This change in rate-limiting step with pH illustrates the danger in the assumption that kcat vs. pH profiles for protease action on substrates with good leaving groups are equivalent to k+3 vs. pH profiles.

Original languageEnglish
Pages (from-to)158-164
Number of pages7
JournalBBA - Enzymology
Volume658
Issue number1
DOIs
Publication statusPublished - Mar 13 1981

Keywords

  • Pre-steady-state kinetics
  • Steady-state kinetics
  • Synthetic substrate
  • Trypsin

ASJC Scopus subject areas

  • Medicine(all)

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