Cataldi de Flombaum M A, Stoppani A O
Mol Biochem Parasitol. 1981 Jul;3(3):143-55. doi: 10.1016/0166-6851(81)90045-1.
Steady-state velocity studies using a substrate regenerating system showed that efrapeptin, citreoviridin and aurovertin inhibit both membrane-bound and soluble mitochondrial ATPase (coupling factor F1) from Trypanosoma cruzi. Maximal inhibitions of ATP hydrolysis produced by efrapeptin and citreoviridin were 100-93%, while the maximal inhibition produced by aurovertin was 40%. Half-maximal inhibitory concentrations decreased in the order citreoviridin greater than aurovertin greater than efrapeptin. Dissociation constants (KD) for the inhibitor-F1 complex were 81 nM (efrapeptin), 6.6 muM (aurovertin) and 40 muM (citreoviridin); KD values for the membrane-bound F1 were 2-4 fold higher than for soluble F1. Representation of efrapeptin inhibition data in the Hill form yielded straight lines (n = 1) while the same representation of citreoviridin inhibition yielded concave down plots. In contrast to the immediate effect of citreoviridin and aurovertin, efrapeptin inhibition was time-dependent. The onset of inhibition, which was pseudo-first-order with respect to efrapeptin, indicated that ATP may promote the binding of efrapeptin to the enzyme. The kinetics of ATP hydrolysis by T. cruzi ATPase as a function MgATP concentration could be explained by the presence of two substrate sites on the enzyme, interacting in such a way that the binding and catalytic events at one site were conformationally linked to the events at the other site, as with the mammalian ATPase. When the antibiotics were assayed at increasing substrate concentrations, efrapeptin produced a linear, mixed-type inhibition whereas citreoviridin produced a parabolic noncompetitive-type inhibition. The aurovertin effect was unusual since the extent of inhibition was greater at high substrate concentrations. Maximal concentrations of all the assayed antibiotics linearized the biphasic double reciprocal plot of control ATPase activity. Comparison of T. cruzi and mammalian F1 responses to the assayed antibiotics revealed the operation of similar inhibition mechanisms but the T. cruzi enzyme was significantly less sensitive to inhibitors than its mammalian counterpart.
利用底物再生系统进行的稳态速度研究表明，埃弗拉菌素、黄绿青霉素和金褐霉素可抑制克氏锥虫的膜结合型和可溶性线粒体ATP酶（偶联因子F1）。埃弗拉菌素和黄绿青霉素对ATP水解的最大抑制率为100% - 93%，而金褐霉素的最大抑制率为40%。半数最大抑制浓度按黄绿青霉素＞金褐霉素＞埃弗拉菌素的顺序降低。抑制剂 - F1复合物的解离常数（KD）分别为81 nM（埃弗拉菌素）、6.6 μM（金褐霉素）和40 μM（黄绿青霉素）；膜结合型F1的KD值比可溶性F1高2 - 4倍。以希尔方程形式表示的埃弗拉菌素抑制数据产生直线（n = 1），而黄绿青霉素抑制的相同表示产生向下凹的图。与黄绿青霉素和金褐霉素的即时作用相反，埃弗拉菌素的抑制作用具有时间依赖性。抑制的起始，相对于埃弗拉菌素为假一级反应，表明ATP可能促进埃弗拉菌素与酶的结合。克氏锥虫ATP酶水解ATP的动力学作为MgATP浓度的函数，可以用酶上存在两个底物位点来解释，这两个位点相互作用，使得一个位点的结合和催化事件在构象上与另一个位点的事件相联系，就像哺乳动物ATP酶一样。当在不断增加的底物浓度下测定抗生素时，埃弗拉菌素产生线性混合型抑制，而黄绿青霉素产生抛物线型非竞争性抑制。金褐霉素的作用不同寻常，因为在高底物浓度下抑制程度更大。所有测定抗生素的最大浓度使对照ATP酶活性的双相双倒数图线性化。克氏锥虫和哺乳动物F1对测定抗生素的反应比较表明，存在相似的抑制机制，但克氏锥虫酶对抑制剂的敏感性明显低于其哺乳动物对应物。
