Sadhu A, Taylor E W
Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637.
J Biol Chem. 1992 Jun 5;267(16):11352-9.
The mechanism of kinesin ATPase has been investigated by transient state kinetic analysis. The results satisfy the scheme [formula: see text] where T, D, and P(i) refer to nucleotide tri- and diphosphate and inorganic phosphate, respectively. The nucleotide-binding steps were measured by the fluorescence enhancement of mant (2'-(3')-O-(N-methylanthraniloyl)-ATP and mant-ADP. The initial rapid equilibrium binding steps (1) and (6) are followed by isomerizations (k2 = 170 +/- 30 s-1 at 20 degrees C, k-5 greater than 100 s-1). The increase in fluorescence is 20-25% larger for K.T** than K.D*. The rate constant of the hydrolysis step k3 is 6-7 s-1. The fluorescence decreases after formation of K.T** at a rate of 7-10 s-1. This change could occur in step 3 or in step 4 if k4 much greater than k3. The value of k4 is larger than 0.1 s-1. The steady state rate is 0.003 s-1 which agrees with the rate of ADP dissociation (k5). Step 5 is rate limiting in the scheme in agreement with the conclusion of Hackney (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318) that ADP dissociation is the rate-limiting step.
驱动蛋白ATP酶的机制已通过瞬态动力学分析进行了研究。结果符合如下反应式[公式：见原文]，其中T、D和P(i)分别指核苷三磷酸、二磷酸和无机磷酸。通过mant（2'-(3')-O-(N-甲基邻氨基苯甲酰基)-ATP和mant-ADP）的荧光增强来测量核苷酸结合步骤。最初的快速平衡结合步骤（1）和（6）之后是异构化（在20℃时k2 = 170±30 s-1，k-5大于100 s-1）。K.T的荧光增加比K.D*大20 - 25%。水解步骤k3的速率常数为6 - 7 s-1。形成K.T后荧光以7 - 10 s-1的速率下降。如果k4远大于k3，这种变化可能发生在步骤3或步骤4中。k4的值大于0.1 s-1。稳态速率为0.003 s-1，这与ADP解离速率（k5）一致。在该反应式中步骤5是限速步骤，这与哈克尼（Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314 - 6318）得出的ADP解离是限速步骤的结论相符。
