Active site serine promotes stabilization of the reactive glutathione thiolate in rat glutathione transferase T2-2. Evidence against proposed sulfatase activity of the corresponding human enzyme.
Ser(11) in rat glutathione transferase T2-2 is important for stabilization of the reactive enzyme-bound glutathione thiolate in the reaction with 1-menaphthyl sulfate. The S11A mutation increased the pK(a) value for the pH dependence of the rate constant for pre-steady-state product formation, from 5.7 to 7.9. This pH dependence is proposed to reflect titration of enzyme-bound glutathione thiol. Further, the mutation lowered the k(cat) value but not because of the impaired stabilization of the glutathione thiolate. In fact, several steps on the reaction pathway were affected by the S11A mutation, and the cause of the decreased k(cat) for the mutant was found to be a slower product release. The data presented here contradict the hypothesis that glutathione transferase T2-2 could act as a sulfatase that is not dependent on Ser(11) for the catalytic activity, as proposed for the corresponding human enzyme (Tan, K.-L., Chelvanayagam, G., Parker, M. W., and Board, P. G. (1996) Biochem. J. 319, 315-321; Rossjohn, J., McKinstry, W. J., Oakley, A. J., Verger, D., Flanagan, J., Chelvanayagam, G., Tan, K.-L., Board, P. G., and Parker, M. W. (1998) Structure 6, 309-322). On the contrary, Ser(11) governs both chemical and physical steps of the catalyzed reaction.
Jemth
P
P
Department of Biochemistry, Uppsala University, Biomedical Center, S-751 23 Uppsala, Sweden.
Mannervik
B
B
D000818:Animals; D020134:Catalytic Domain; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007231:Infant, Newborn; D007700:Kinetics; D008623:Mercaptoethanol / Q000378:metabolism; D008956:Models, Chemical; D009154:Mutation; D009578:Nitrobenzenes / Q000378:metabolism; D051381:Rats; D012694:Serine; D013429:Sulfatases / Q000378:metabolism; D014783:Viscosity
D009578:Nitrobenzenes; C008347:S-(1-menaphthyl)glutathione; C027562:4-nitrobenzyl chloride; D012694:Serine; D008623:Mercaptoethanol; C491107:GSTT2 protein, human; D005982:Glutathione Transferase; D013429:Sulfatases; D005978:Glutathione