Reynafarje B D, Pedersen P L
Department of Biological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205-2185, USA.
J Biol Chem. 1996 Dec 20;271(51):32546-50. doi: 10.1074/jbc.271.51.32546.
Conditions have been reported under which the F1 moiety of bovine heart ATP synthase catalyzes the hydrolysis of ATP by an apparently cooperative mechanism in which the slow rate of hydrolysis at a single catalytic site (unisite catalysis) is enhanced more than 10(6)-fold when ATP is added in excess to occupy one or both of the other two catalytic sites (multisite catalysis) (Cross, R. L., Grubmeyer, C., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12101-12105). In the novel studies reported here, and in contrast to the earlier report, we have (a) monitored the kinetics of ATP hydrolysis of F1 by using nucleotide-depleted preparations and a highly sensitive chemiluminescent assay; (b) followed the reaction immediately upon addition of F1 to ATP, rather than after prior incubation with ATP; and (c) used a reaction medium with Pi as the only buffer. The following observations were noted. First, regardless of the source of enzyme, bovine or rat, and catalytic conditions (unisite or multisite), the rates of hydrolysis depend on ATP concentration to the first power. Second, the first order rate constant for ATP hydrolysis remains relatively constant under both unisite and multisite conditions declining only slightly at high ATP concentration. Third, the initial rates of ATP hydrolysis exhibit Michaelis-Menten kinetic behavior with a single Vmax exceeding 100 micromol of ATP hydrolyzed per min/mg of F1 (turnover number = 635 s-1) and a single Km for ATP of about 57 microM. Finally, the reaction is inhibited markedly by low concentrations of ADP. It is concluded that, under the conditions described here, all catalytic sites that participate in the hydrolysis of ATP within the F1 moiety of mitochondrial ATP synthase function in a kinetically equivalent manner.
据报道，在某些条件下，牛心ATP合酶的F1部分通过一种明显的协同机制催化ATP水解，即当过量添加ATP以占据另外两个催化位点中的一个或两个时（多位点催化），单个催化位点的缓慢水解速率（单一位点催化）会提高超过10^6倍（克罗斯、R.L.、格鲁布迈尔、C.和佩内夫斯基、H.S.（1982年）《生物化学杂志》257，12101 - 12105）。在本文报道的新研究中，与早期报告不同的是，我们（a）使用核苷酸耗尽的制剂和高灵敏度的化学发光测定法监测F1的ATP水解动力学；（b）在将F1添加到ATP后立即跟踪反应，而不是在与ATP预先孵育之后；（c）使用以Pi作为唯一缓冲剂的反应介质。有以下观察结果。首先，无论酶的来源是牛还是大鼠，以及催化条件（单一位点或多位点）如何，水解速率都与ATP浓度的一次方相关。其次，在单一位点和多位点条件下，ATP水解的一级速率常数保持相对恒定，仅在高ATP浓度下略有下降。第三，ATP水解的初始速率呈现米氏动力学行为，单个Vmax超过每分钟每毫克F1水解100微摩尔ATP（周转数 = 635 s^-1），ATP的单个Km约为57 microM。最后，该反应受到低浓度ADP的显著抑制。结论是，在此处描述的条件下，线粒体ATP合酶F1部分中参与ATP水解的所有催化位点在动力学上以等效方式起作用。
