GSTB1-1 from Proteus mirabilis: a snapshot of an enzyme in the evolutionary pathway from a redox enzyme to a conjugating enzyme.

The native form of the bacterial glutathione transfer- ase B1-1 (EC 2.5.1.18) is characterized by one glutathi- one (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 nor- mally performed by GSTs. Stopped-flow and spectro- scopic 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 pKa 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 in- duces a more active deprotonation of the substrate with an apparent pKa of 5.2 0.1. Apparently, the absence of the mixed disulfide does not affect kcat and Km 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 se- quential mechanism whereas a rapid-equilibrium ran- dom sequential mechanism is adopted by GSTB1-1red. Remarkably, GSTB1-1ox and GSTB1-1red are equally able to catalyze a glutaredoxin-like catalysis using cys- teine 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 ac- tivity completely. It appears that GSTB1-1 behaves like an “intermediate enzyme” between the thiol-disulfide oxidoreductase and the GST superfamilies.