Biosca J A, Eisenberg E
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1990 Jun 25;265(18):10221-5.
We previously studied the equilibrium binding of ADP, adenyl-5'-yl imidodiphosphate (AMP-PNP), and inorganic pyrophosphate (PPi) to actomyosin-subfragment 1 (acto.S-1) and found that AMP-PNP and PPi bind considerably more weakly to acto.S-1 than does ADP. In this study, we investigated the pre-steady-state kinetics of the binding of AMP-PNP and PPi to acto.S-1 and of S-1.AMP-PNP and S-1.PPi to actin to determine if the pre-steady-state kinetic data are consistent with our previous equilibrium data. We find that the kinetic data are consistent with the equilibrium data and agree with a model in which acto.S-1 forms a collision intermediate with the ATP analog, followed by a slower conformational change to a ternary complex that rapidly dissociates into actin and the S-1.ATP analog. Although this scheme fits the AMP-PNP as well as the PPi data, we find that the isomerization of the collision intermediate to the ternary complex is approximately 10 times faster in the presence of PPi than in the presence of AMP-PNP, which is consistent with previous physiological studies (Schoenberg, M., and Eisenberg, E. (1985) Biophys. J. 48, 863-872).
我们之前研究了ADP、腺苷-5'-亚基咪唑二磷酸（AMP-PNP）和无机焦磷酸（PPi）与肌动球蛋白亚片段1（肌动蛋白.S-1）的平衡结合，发现AMP-PNP和PPi与肌动蛋白.S-1的结合比ADP弱得多。在本研究中，我们研究了AMP-PNP和PPi与肌动蛋白.S-1以及S-1.AMP-PNP和S-1.PPi与肌动蛋白结合的预稳态动力学，以确定预稳态动力学数据是否与我们之前的平衡数据一致。我们发现动力学数据与平衡数据一致，并且与一个模型相符，在该模型中，肌动蛋白.S-1与ATP类似物形成碰撞中间体，随后发生较慢的构象变化形成三元复合物，该复合物迅速解离为肌动蛋白和S-1.ATP类似物。尽管该方案适用于AMP-PNP以及PPi的数据，但我们发现，在PPi存在下，碰撞中间体异构化为三元复合物的速度比在AMP-PNP存在下快约10倍，这与之前的生理学研究结果一致（Schoenberg, M., and Eisenberg, E. (1985) Biophys. J. 48, 863 - 872）。
