Catalytic site cooperativity of beef heart mitochondrial F1 adenosine triphosphatase. Correlations of initial velocity, bound intermediate, and oxygen exchange measurements with an alternating three-site model.
Catalytic site cooperativity of beef heart mitochondrial F1 adenosine triphosphatase. Correlations of initial velocity, bound intermediate, and oxygen exchange measurements with an alternating three-site model.
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DOI：
10.1016/s0021-9258(18)33672-x
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发表时间：
1982-10
期刊：
影响因子：
--
通讯作者：
M. Gresser;J. A. Myers;P. Boyer
中科院分区：
文献类型：
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作者：
M. Gresser;J. A. Myers;P. Boyer
以前对牛心线粒体F1-ATPase的研究表明，一个催化部位的底物(ATP)结合加速了另一个催化部位的产物(ADP+PI)的释放。本文报道的组氨酸速度研究与这一机制相一致，显示出两相速率响应，表观K值分别为250和1.7 PM，V值分别为54和2.2pmol/min/mg。此外，所提出的机制导致了对F1 ATPase的一个重要的、以前未被检验的诊断预测，即在底物浓度远低于给出半最大速度的底物浓度时，在周转过程中将出现高水平的酶结合产物。这一预测已被证明，大多数F1 ATPase分子在低于半最大速度时的浓度时，将产物ADP保留在催化位置。该机制还预测了底物结合的负协同性，产物形成速度较慢，直到所有催化位置都被填满。预测的底物结合模式被本文报道的1催化位点/Fl ATP-酶饱和而仅存在约1，UM的ATP所证实。这个单一占据的位置显示出缓慢的催化翻转，在1p~ATP处形成的PI的大部分氧已与水交换。当ATP浓度增加到10-20 PM时，这种氧交换明显减少，但仍远低于半最大速度所需的ATP浓度。这些结果既不能通过独立地点机制也不能通过相互作用的两地点机制来令人满意地相互关联。提出了一种简单的相互作用三位点机制，令人满意地关联了所有三组结果。过去几年来，有足够的证据支持这样的观点，即ATP合成酶复合体通过反应物结合中的能量连接变化(结合-改变机制)合成ATP。1和2用于概述这一证据)。这些与能量相关的
Previous studies with beef heart mitochondrial F1 ATPase suggest a mechanism in which binding of substrate (ATP) at one catdytic site accelerates the release of products (ADP+ Pi) from a separate catalytic site. hitid velocity studies reported here, in harmony with this mechanism, show a biphasic rate response with apparent K,,, values of 250 and 1.7 PM and V,, values of 54 and 2.2 pmol/min/mg. Further, the suggested mechanism leads to an important and previously untested diagnostic prediction for F1 ATPase, namely that high levels of enzyme-bound product will be present during turnover at substrate concentrations well below those which give half-maximal velocity. This prediction has been verified by the demonstration that most F1 ATPase molecules retain product ADP at a catalytic site when hydrolyzing ATP at concentrations less than ‘hoo of that for half-maximal velocity. The mechanism also predicts a negative cooperativity of substrate binding, with a slow rate of product formation until all catalytic sites are filIed. The predicted substrate binding pattern is substantiated by the demonstration reported herein that 1 catalytic site/Fl ATP-ase becomes saturated with only about 1, UM ATP present. This singly occupied site shows slow catalytic turn-over, and most of the oxygens of Pi formed at 1 p~ ATP have been exchanged with water. Pronounced decrease in this oxygen exchange occurs when the ATP concentration is increased to 10-20 PM, which is still much lower than the concentration of ATP required for halfmaximal velocity. These results cannot be satisfactorily correlated by either independent-site mechanisms or by interacting two-site mechanisms. A simple interact-ing three-site mechanism is presented that satisfactorily correlates all three sets of results.Considerable evidence has accrued over the past several years supporting the view that ATP synthesis by the ATP synthase complex occurs through energy-linked changes in reactant binding (the “binding-change mechanism”; see Refs. 1 and 2 for an outline of this evidence). These energy-linked