Kinetic mechanism of octopus hepatopancreatic glutathione transferase in reverse micelles.

Abstract

Octopus glutathione transferase (GST) was enzymically active in aerosol-OT [sodium bis-(2-ethylhexyl)sulphosuccinate]/iso-octane reverse micelles albeit with lowered catalytic constant (kcat). The enzyme reaction rate was found to be dependent on the [H2O]/[surfactant] ratio (omega(o)) of the system with maximum rate observed at omega(o) 13.88, which corresponded to vesicles with a core volume of 64 nm3. According to the physical examinations, a vesicle of this size is barely large enough to accommodate a monomeric enzyme subunit. Dissociation of the enzyme in reverse micelles was confirmed by cross-linking of the associated subunits with glutaraldehyde and separation of the monomers and dimers with electrophoresis in the presence of SDS. The kinetic properties of the enzyme were investigated by steady-state kinetic analysis. Both GSH and 1-chloro-2,4-dinitrobenzene (CDNB) showed substrate inhibition and the Michaelis constant for CDNB was increased by 36-fold to 11.05 mM in reverse micelles. Results on the initial-velocity and product-inhibition studies indicate that the octopus GST conforms to a steady-state sequential random Bi Bi mechanism. The results from a log kcat versus pH plot suggest that amino acid residues with pKa values of 6.56 0.07 and 8.81 0.17 should be deprotonated to give optimum catalytic function. In contrast, the amino acid residue with a pKa value of 9.69 0.16 in aqueous solution had to be protonated for the reaction to proceed. We propose that the pKa1 (6.56) is that for the enzyme-bound GSH, which has a pKa value lowered by 1.40-1.54 pH units compared with that of free GSH in reverse micelles. The most probable candidate for the observed pKa2 (8.81) is Tyr7 of GST. The pKa of Tyr7 is 0.88 pH unit lower than that in aqueous solution and is about 2 pH units below the normal tyrosine. This tyrosyl residue may act as a base catalyst facilitating the dissociation of enzyme-bound GSH. The possible interaction of GST with plasma membrane in vivo is discussed.