title: GSTB1-1 from Proteus mirabilis: a snapshot of an enzyme in the evolutionary pathway from a redox enzyme to a conjugating enzyme.
abstract: The native form of the bacterial glutathione transferase B1-1 (EC ) is characterized by one glutathione (GSH) molecule covalently linked to Cys-10. This peculiar disulfide, only found in the Beta and Omega class glutathione S-transferases (GSTs) but absent in all other GSTs, prompts questions about its role and how GSH can be activated and utilized in the reaction normally performed by GSTs. Stopped-flow and spectroscopic experiments suggest that, in the native enzyme (GSTB1-1ox), a second GSH molecule is present, albeit transiently, in the active site. This second GSH binds to the enzyme through a bimolecular interaction followed by a fast thiol-disulfide exchange with the covalently bound GSH. The apparent pK(a) of the non-covalently bound GSH is lowered from 9.0 to 6.4 +/- 0.2 in similar fashion to other GSTs. The reduced form of GSTB1-1 (GSTB1-1red) binds GSH 100-fold faster and also induces a more active deprotonation of the substrate with an apparent pK(a) of 5.2 +/- 0.1. Apparently, the absence of the mixed disulfide does not affect k(cat) and K(m) values in the GST conjugation activity, which is rate-limited by the chemical step both in GSTB1-1red and in GSTB1-1ox. However, GSTB1-1ox follows a steady-state random sequential mechanism whereas a rapid-equilibrium random sequential mechanism is adopted by GSTB1-1red. Remarkably, GSTB1-1ox and GSTB1-1red are equally able to catalyze a glutaredoxin-like catalysis using cysteine S-sulfate and hydroxyethyl disulfide as substrates. Cys-10 is an essential residue in this redox activity, and its replacement by alanine abolishes this enzymatic activity completely. It appears that GSTB1-1 behaves like an "intermediate enzyme" between the thiol-disulfide oxidoreductase and the GST superfamilies.
authors.affiliation: Department of Biology, University of Rome, Tor Vergata, 00133 Rome, Italy.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Caccuri
authors.forename: Giovanni
authors.initials: G
authors.lastname: Antonini
authors.forename: Nerino
authors.initials: N
authors.lastname: Allocati
authors.forename: Carmine
authors.initials: C
authors.lastname: Di Ilio
authors.forename: Francesca
authors.initials: F
authors.lastname: De Maria
authors.forename: Federica
authors.initials: F
authors.lastname: Innocenti
authors.forename: Michael W
authors.initials: MW
authors.lastname: Parker
authors.forename: Michele
authors.initials: M
authors.lastname: Masulli
authors.forename: Mario
authors.initials: M
authors.lastname: Lo Bello
authors.forename: Paola
authors.initials: P
authors.lastname: Turella
authors.forename: Giorgio
authors.initials: G
authors.lastname: Federici
authors.forename: Giorgio
authors.initials: G
authors.lastname: Ricci
mesh_terms: D002384:Catalysis; D019143:Evolution, Molecular; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D011485:Protein Binding; D011487:Protein Conformation; D011490:Protein Disulfide Reductase (Glutathione) / Q000378:metabolism; D011506:Proteins / Q000737:chemistry; D011513:Proteus mirabilis / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D054477:Glutaredoxins; D011506:Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D005982:Glutathione Transferase; D005978:Glutathione