Properties and Utility of the Peculiar Mixed Disulfide in the Bacterial Glutathione Transferase B1-1†
- Anna Maria Caccuri
- ,
- Giovanni Antonini
- ,
- Nerino Allocati
- ,
- Carmine Di Ilio
- ,
- Federica Innocenti
- ,
- Francesca De Maria
- ,
- Michael W. Parker
- ,
- Michele Masulli
- ,
- Francesca Polizio
- ,
- Giorgio Federici
- , and
- Giorgio Ricci
Abstract
Bacterial glutathione transferases appear to represent an evolutionary link between the thiol:disulfide oxidoreductase and glutathione transferase superfamilies. In particular, the observation of a mixed disulfide in the active site of Proteus mirabilis glutathione transferase B1-1 is a feature that links the two families. This peculiar mixed disulfide between Cys10 and one GSH molecule has been studied by means of ESR spectroscopy, stopped-flow kinetic analysis, radiochemistry, and site-directed mutagenesis. This disulfide can be reduced by dithiothreitol but even a thousand molar excess of GSH is poorly effective due to an unfavorable equilibrium constant of the redox reaction (Keq = 2 × 10-4). Although Cys10 is partially buried in the crystal structure, in solution it reacts with several thiol reagents at a higher or comparable rate than that shown by the free cysteine. Kinetics of the reaction of Cys10 with 4,4‘-dithiodipyridine at variable pH values is consistent with a pKa of 8.0 ± 0.1 for this residue, a value about 1 unit lower than that of the free cysteine. The 4,4‘-dithiodipyridine-modified enzyme reacts with GSH in a two-step mechanism involving a fast precomplex formation, followed by a slower chemical step. The natural Cys10-GSH mixed disulfide exchanges rapidly with free [3H]GSH in a futile redox cycle in which the bound GSH is continuously replaced by the external GSH. Our data suggest that the active site of the bacterial enzyme has intermediate properties between those of the recently evolved glutathione transferases and those of the thiol:disulfide oxidoreductase superfamily.
†
G.R., C.D.I., and N.A. were partially supported by MURST PRIN2000. G.R. and G.A. were partially supported by the National Research Council of Italy (Target Project on Biotechnology).
‡
University of Rome Tor Vergata.
§
University of L'Aquila and University of Rome Three.
‖
University of Chieti G. D'Annunzio.
⊥
St. Vincent's Institute of Medical Research.
#
Children's Hospital IRCCS Bambin Gesu.
*
To whom correspondence should be addressed. Tel: +39 0672594379. Fax: +39 0672594311. E-mail: [email protected].
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