title: 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.
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 kcat 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.
authors.affiliation: Department of Biology, University of Rome "Tor Vergata," 00133 Rome, Italy.
authors.forename: A M
authors.initials: AM
authors.lastname: Caccuri
authors.forename: P
authors.initials: P
authors.lastname: Ascenzi
authors.forename: G
authors.initials: G
authors.lastname: Antonini
authors.forename: M W
authors.initials: MW
authors.lastname: Parker
authors.forename: A J
authors.initials: AJ
authors.lastname: Oakley
authors.forename: E
authors.initials: E
authors.lastname: Chiessi
authors.forename: M
authors.initials: M
authors.lastname: Nuccetelli
authors.forename: A
authors.initials: A
authors.lastname: Battistoni
authors.forename: A
authors.initials: A
authors.lastname: Bellizia
authors.forename: G
authors.initials: G
authors.lastname: Ricci
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism*; D001665:Binding Sites; D004137:Dinitrochlorobenzene / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D008961:Models, Structural; D008962:Models, Theoretical; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D004137:Dinitrochlorobenzene; D007527:Isoenzymes; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D009327:4-Chloro-7-nitrobenzofurazan; D005978:Glutathione