Structural flexibility modulates the activity of human glutathione transferase P1-1: Influence of a poor co-substrate on dynamics and kinetics of human glutathione transferase

Anna Maria Caccuri, Paolo Ascenzi, Giovanni Antonini, Michael W. Parker, Aaron J. Oakley, Ester Chiessi, Marzia Nuccetelli, Andrea Battistoni, Anna Bellizia, Giorgio Ricci

Research output: Contribution to journalArticlepeer-review

Abstract

Presteady-state and steady-state kinetics of human glutathione transferase P1-1 (EC 2.5.1.18) have been studied at pH 5.0 by using 7- chloro-4-nitrobenzo-2-oxa-1,3-diazole, a poor co-substrate for this isoenzyme. Steady-state kinetics fits well with the simplest rapid equilibrium random sequential bi-bi mechanism and reveals a strong intrasubunit synergistic modulation between the GSH-binding site (G-site) and the hydrophobic binding site for the co-substrate (H-site); the affinity of the G-site for GSH increases about 30 times at saturating co-substrate and vice versa. Presteady-state experiments and thermodynamic data indicate that the rate-limiting step is a physical event and, probably, a structural transition of the ternary complex. Similar to that observed with 1-chloro- 2,4-dinitrobenzene (Ricci, G. Caccuri, A. M., Lo Bello, M., Rosato, N., Mei, G., Nicotra, M., Chiessi, E., Mazzetti, A. P., and Federici, G. (1996) J. Biol. Chem. 271, 16187-16192), this event may be related to the frequency of enzyme motions. The observed low, viscosity-independent k(cat) value suggests that these motions are slow and diffusion-independent for an increased internal viscosity. In fact, molecular modeling suggests that the hydroxyl group of Tyr-108, which resides in helix 4, may be in hydrogen bonding distance of the oxygen atom of this new substrate, thus yielding a less flexible H-site. This effect might be transmitted to the G-site via helix 4. In addition, a new homotropic behavior exhibited by 7-chloro-4-nitrobenzo-2- oxa-1,3-diazole is found in Cys-47 mutants revealing a structural intersubunit communication between the two H-sites.

Original languageEnglish
Pages (from-to)16193-16198
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number27
DOIs
Publication statusPublished - 1996

ASJC Scopus subject areas

  • Biochemistry

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