Harris D A
Department of Biochemistry, University of Oxford, U.K.
Biochim Biophys Acta. 1989 May 8;974(2):156-62. doi: 10.1016/s0005-2728(89)80368-8.
(1) The hydrolytic activity of the isolated mitochondrial ATPase (F1) is strongly inhibited by azide. However, at very low ATP concentration (1 microM or less), no inhibition by azide is observed. (2) The azide-insensitive ATPase activity represents a high-affinity, low-capacity mode of turnover of F1. This is identified with the low Km, low Vmax component seen in steady-state kinetic studies in the absence of azide. (3) The azide-insensitive ATPase activity shows simple Michaelis-Menten kinetics, with Km = 3.2 microM, and Vmax = 1.1 mumol/min per mg (6 s-1). It is unaffected by anions such as sulphite, or by increasing pH in the range 7 to 8, both of which stimulate the maximal activity of F1. (4) Both the azide-insensitive and azide-sensitive components of F1-ATPase activity are equally inhibited by labelling the enzyme with 7-chloro-4-nitrobenzofurazan, by binding the natural inhibitor protein, or by cold denaturation of the enzyme. (5) It is concluded that azide-insensitive ATP hydrolysis represents catalysis by F1 involving a single catalytic site, and that azide acts by abolishing intersubunit cooperativity between the three catalytic sites of F1. Azide-sensitivity is thus a useful probe for events which affect the active site of F1 directly.
(1) 分离出的线粒体ATP酶（F1）的水解活性受到叠氮化物的强烈抑制。然而，在非常低的ATP浓度（1微摩尔或更低）下，未观察到叠氮化物的抑制作用。(2) 对叠氮化物不敏感的ATP酶活性代表F1的一种高亲和力、低容量的周转模式。这与在无叠氮化物的稳态动力学研究中看到的低Km、低Vmax组分一致。(3) 对叠氮化物不敏感的ATP酶活性表现出简单的米氏动力学，Km = 3.2微摩尔，Vmax = 1.1微摩尔/分钟·毫克（6秒⁻¹）。它不受亚硫酸盐等阴离子的影响，也不受pH值在7至8范围内升高的影响，而这两种情况都会刺激F1的最大活性。(4) 通过用7-氯-4-硝基苯并呋喃标记酶、结合天然抑制蛋白或使酶冷变性，F1-ATP酶活性的对叠氮化物不敏感和对叠氮化物敏感的组分均受到同等程度的抑制。(5) 得出的结论是，对叠氮化物不敏感的ATP水解代表F1通过单个催化位点进行的催化作用，并且叠氮化物通过消除F1三个催化位点之间的亚基间协同作用而起作用。因此，叠氮化物敏感性是直接影响F1活性位点事件的有用探针。
