KINETIC MECHANISM OF OCTOPUS HEPATOPANCREATIC GLUTATHIONE TRANSFERASEIN REVERSE MICELLES

Octopus glutathione transferase (GST) was enzymically active in rosol- OT [sodium bis-(2-ethylhexyl)sulphosuccinate] /iso-octane reverse mice lles albeit with lowered catalytic constant (k(cat)). The enzyme react ion rate was found to be dependent on the [H2O]/[surfactant] ratio (om ega(0)) of the system with maximum rate observed at omega, 13.88, whic h corresponded to vesicles with a core volume of 64 nm(3). According t o the physical examinations, a vesicle of this size is barely large en ough to accommodate a monomeric enzyme subunit. Dissociation of the en zyme in reverse micelles was confirmed by cross-linking ofthe associat ed subunits with glutaraldehyde and separation of the monomers and dim ers with electrophoresis in the presence of SDS. The kinetic propertie s of the enzyme were investigated by steady-state kinetic analysis. Bo th GSH and 1-chloro-2,4-dinitrobenzene (CDNB) showed substrate inhibit ion and the Michaelis constant for CDNB was increased by 36-fold to 11 .05 mM in reverse micelles. Results on the initial-velocity and produc t-inhibition studies indicate that the octopus GST conforms to a stead y-state sequential random Bi Bi mechanism. The results from a log k(ca t) versus pH plot suggest that amino acid residues with pK(a) values o f 6.56+/-0.07 and 8.81+/-0.17 should be deprotonated to give optimum c atalytic function. In contrast, the amino acid residue with a pK(a) va lue of 9.69+/-0.16 in aqueous solution had to be protonated for the re action to proceed. We propose that the pK(a1) (6.56) is that for the e nzyme-bound GSH, which has a pK(a) 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 pK(a2) (8.81) is Tyr(7) of GST. The pK(a) of Tyr(7) is 0.88 pH unit lower than that in aqueous solution and is a bout 2 pH units below the normal tyrosine. This tyrosyl residue may ac t as a base catalyst facilitating the dissociation of enzyme-bound GSH . The possible interaction of GST with plasma membrane in vivo is disc ussed.