Are the Steady State Kinetics of Glutathione Transferase Always Dependent on the Deprotonation of the Bound Glutathione?. New Insights in the Kinetic Mechanism of GST-P-1-1

A. M. Caccuri, P. Ascenzi, M. Lobello, G. Federici, A. Battistoni, P. Mazzetti, G. Ricci

Research output: Contribution to journalArticlepeer-review

Abstract

Steady state kinetics measurements performed on human placenta glutathione transferase (GST P 1-1), utilizing 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) as co-substrate, show that the kcat value (≅1.2 s-1) is pH-independent between pH 4.0 and 7.0 and is scarcely affected by the nature of the leaving group. The pH profile of kcat/KNBD-Cl m suggests a pKa ≥ 6.0 for GSH bound to the enzyme. Pre-steady state experiments demonstrate the presence of a burst-phase in which the conjugation product (or the σ-complex intermediate) accumulates in an amount stoichiometric to the GST active site concentration. These results indicate that the steady state kinetics of GST P 1-1 with NBD-Cl are independent of the deprotonation of the bound GSH between pH 4.0 and 7.0 because the rate-limiting step is the product release. The occurrence of a fast enzymatic conjugation of GSH with a number of poor substrates or even electrophilic inhibitors of GST, mainly performed in a single turnover reaction, may reveal a further detoxicating role of GST.

Original languageEnglish
Pages (from-to)1428-1434
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume200
Issue number3
DOIs
Publication statusPublished - May 15 1994

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

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