Kinetic characterization of the endogenous glutathione transferase activity of octopus lens S-crystallin.
The kinetic mechanism of the endogenous glutathione transferase (GST) activity of octopus S-crystallin was investigated by steady-state kinetics. Biphasic double-reciprocal plots were obtained for both glutathione and the hydrophobic substrate 1-chloro-2,4-dinitrobenzene (CDNB). Substrate inhibition was observed only for CDNB with Ksi value of 29.7 +/- 0.01 mM. The catalytic constant for S-crystallin was three orders of magnitude smaller than that for the digestive gland GST of the same species. The initial-velocity studies indicated that the enzyme reaction might conform to a steady-state random Bi-Bi kinetic mechanism, being similar to the reaction of GST from other sources. The pH-rate profiles also suggest that the same chemical mechanism for the nucleophilic aromatic substitution between GSH and CDNB was employed for S-crystallin. The interaction of Tyr7 with the bound GSH lowered the pKa value of the sulfhydryl group of GSH to 6.82-6.85, which is 2.32-2.35 pH unit smaller than that found in aqueous solution. This lowering of pKa value produces the thiolate anion of GSH, a better nucleophile to attack the ipso carbon of CDNB, resulting in formation of Meisenheimer complex intermediate. Removing the chloride ion from this intermediate complex produces the conjugate product. Using the method devised by Wang and Srivastava (Anal. Biochem. 216, 15-26, 1994), the functional unit of the dimeric S-crystallin was estimated to be a monomer. The possible biological implications of the endogenous detoxification ability of cephalopods S-crystallin are discussed.
Tang
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SS
Graduate Institutes of Life Sciences and Biochemistry, National Defense Medical Center, Taipei, Taiwan.
Chang
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GG
D000818:Animals; D001665:Binding Sites; D003459:Crystallins / Q000378:metabolism*; D004137:Dinitrochlorobenzene / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D049831:Octopodiformes
D003459:Crystallins; D004137:Dinitrochlorobenzene; D005982:Glutathione Transferase; D005978:Glutathione