Hays J B, Sussman M L
Biochim Biophys Acta. 1976 Aug 16;443(2):267-83. doi: 10.1016/0005-2736(76)90509-5.
Tosyl galactosides, previously shown to be potent reversible dead-end inhibitors of the membrane-bound Enzyme IIlac of the lactose phosphotransferase system of Staphylococcus aureus, were used for an investigation of the kinetic mechanism of the sugar phosphorylation/transport reaction catalyzed by this enzyme: phospho-Factor IIIlac&sugar Enzyme IIlac lead to Factor IIIlac&sugar phosphate. Inhibition of Enzyme IIlac was studied in three different systems. Washed membranes, and washed membranes in the presence of 0.1% Triton X-100 were used for phosphorylation experiments, and whole cells were used for transport studies. When washed membranes were used to supply Enzyme IIlac, inhibition of phosphorylation by tosyl galactoside was linear non-competitive against both the sugar and phospho-Factor IIIlac substrates, with an apparent Ki of about 0.5 mM. This Ki decreased with increasing Factor IIIlac concentration. In the presence of 0.1% Triton X-100, the phosphorylation reaction was stimulated; under these conditions the inhibition became strictly competitive against sugar, and completely uncompetitive against phospho-Factor IIIlac. Apparently washed membranes can catalyze phosphorylation both via a reaction sequence in which sugar binds first and via one in which phospho-Factor IIIlac binds first, but in the presence of 0.1% Triton the reaction does not occur by the former sequence. The inability of bound phospho-Factor IIIlac to hinder the binding of tosyl galactosides suggests that the initial binding sites of the two substrates of Enzyme IIlac are separated by at least the distance of the tosyl moiety. Radioactive methyl 6-O-(p-toluenesulfonyl) beta-galactoside was not converted into a phosphorylated product in the reaction mixtures, i.e. it is a true dead-end inhibitor. Inhibition of beta-galactoside transport into whole cells by tosyl galactosides was competitive, with an apparent Ki of 5-10 mM, an order of magnitude higher than the Ki for inhibition of phosphorylation by membrane preparations. This result suggest that a significant level of unphosphorylated phospho-Factor IIIlac is present inside the cells, or that cellular levels of this compound are considerably lower than those used for in vitro sugar phosphorylation assays. Radioactive tosyl galactoside inhibitor was not transported into whole cells.
甲苯磺酰半乳糖苷先前已被证明是金黄色葡萄球菌乳糖磷酸转移酶系统中膜结合的酶IIlac的有效可逆性终产物抑制剂，被用于研究该酶催化的糖磷酸化/转运反应的动力学机制：磷酸化因子IIIlac&糖 + 酶IIlac → 因子IIIlac&磷酸糖。在三种不同体系中研究了酶IIlac的抑制作用。洗涤过的膜以及存在0.1% Triton X - 100的洗涤过的膜用于磷酸化实验，完整细胞用于转运研究。当使用洗涤过的膜来提供酶IIlac时，甲苯磺酰半乳糖苷对磷酸化的抑制作用对糖和磷酸化因子IIIlac底物均呈线性非竞争性，表观Ki约为0.5 mM。该Ki随因子IIIlac浓度增加而降低。在存在0.1% Triton X - 100的情况下，磷酸化反应受到刺激；在这些条件下，抑制作用对糖变为严格竞争性，对磷酸化因子IIIlac则完全非竞争性。显然，洗涤过的膜可以通过糖先结合的反应序列以及磷酸化因子IIIlac先结合的反应序列催化磷酸化，但在存在0.1% Triton的情况下，反应不会通过前一种序列发生。结合的磷酸化因子IIIlac无法阻碍甲苯磺酰半乳糖苷的结合，这表明酶IIlac的两种底物的初始结合位点至少相隔甲苯磺酰部分的距离。放射性甲基6 - O -（对甲苯磺酰基）β - 半乳糖苷在反应混合物中未转化为磷酸化产物，即它是一种真正的终产物抑制剂。甲苯磺酰半乳糖苷对完整细胞中β - 半乳糖苷转运的抑制作用是竞争性的，表观Ki为5 - 10 mM，比膜制剂抑制磷酸化的Ki高一个数量级。这一结果表明细胞内存在显著水平的未磷酸化的磷酸化因子IIIlac，或者该化合物的细胞水平远低于体外糖磷酸化测定中所使用的水平。放射性甲苯磺酰半乳糖苷抑制剂未转运进入完整细胞。
