Tang S S, Chang G G
Graduate Institutes of Life Sciences and Biochemistry, National Defence Medical Centre, Taipei, Taiwan, Republic of China.
Biochem J. 1996 Apr 15;315 ( Pt 2)(Pt 2):599-606. doi: 10.1042/bj3150599.
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.
章鱼谷胱甘肽转移酶（GST）在气溶胶-OT[双（2-乙基己基）磺基琥珀酸钠]/异辛烷反胶束中具有酶活性，尽管催化常数（kcat）有所降低。发现酶反应速率取决于体系的[水]/[表面活性剂]比率（ωo），在ωo为13.88时观察到最大反应速率，这对应于核心体积为64nm3的囊泡。根据物理检测，这种大小的囊泡勉强足够大以容纳一个单体酶亚基。通过用戊二醛交联相关亚基并在SDS存在下用电泳分离单体和二聚体，证实了酶在反胶束中的解离。通过稳态动力学分析研究了该酶的动力学性质。谷胱甘肽（GSH）和1-氯-2,4-二硝基苯（CDNB）均表现出底物抑制，并且在反胶束中CDNB的米氏常数增加了36倍至11.05mM。初始速度和产物抑制研究的结果表明，章鱼GST符合稳态有序随机双底物双产物机制。log kcat对pH作图的结果表明，pKa值为6.56±0.07和8.81±0.17的氨基酸残基应去质子化以提供最佳催化功能。相比之下，水溶液中pKa值为9.69±0.16的氨基酸残基必须质子化才能使反应进行。我们提出pKa1（6.56）是酶结合的GSH的pKa值，与反胶束中游离GSH的pKa值相比，其降低了1.40-1.54个pH单位。观察到的pKa2（8.81）最可能的候选者是GST的Tyr7。Tyr7的pKa比水溶液中的低0.88个pH单位，比正常酪氨酸低约2个pH单位。这个酪氨酸残基可能作为碱催化剂促进酶结合的GSH的解离。讨论了GST在体内与质膜的可能相互作用。
