Fast product formation and slow product release are important features in a hysteretic reaction mechanism of glutathione transferase T2-2.
The reaction mechanism of rat glutathione transferase T2-2 has been studied using pre-steady-state and steady-state kinetics. Several parts of the catalytic cycle including binding of substrates, product formation, and product release were investigated. Under saturating conditions, a two-step product release was found to be rate limiting in the enzyme-catalyzed reactions between the nucleophilic substrate glutathione and either of the two electrophilic substrates 1-menaphthyl sulfate and 4-nitrobenzyl chloride. The rate constant for pre-steady-state product formation on rat glutathione transferase T2-2 has an observed pK(a) value of 5.7 apparently due to ionization of the sulfhydryl group of glutathione. This rate constant is approximately 2 orders of magnitude higher than k(cat) at pH values of >6. It can be predicted from the pH dependence that product formation would be the sole rate-limiting step at pH values of <3. A hysteretic mechanism of rGST T2-2 is proposed based on a slow conformational transition detected in pre-steady-state displacement experiments.
Jemth
P
P
Department of Biochemistry, Uppsala University, Biomedical Center, Sweden.
Mannervik
B
B
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009281:Naphthalenes / Q000737:chemistry; D009578:Nitrobenzenes / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013053:Spectrophotometry; D013379:Substrate Specificity; D013997:Time Factors
D009281:Naphthalenes; D009578:Nitrobenzenes; D011994:Recombinant Proteins; C027562:4-nitrobenzyl chloride; C491107:GSTT2 protein, human; D005982:Glutathione Transferase; D005978:Glutathione; C008348:1-menaphthyl sulfate