Ma Y Z, Taylor E W
Department of Molecular Genetics and Cell Biology, The University of Chicago, Illinois 60637, USA.
J Biol Chem. 1997 Jan 10;272(2):717-23. doi: 10.1074/jbc.272.2.717.
The kinetic mechanism is analyzed for a monomeric human kinesin construct K332. In the absence of microtubules, the rate constants of the ATPase cycle are very similar to dimeric human kinesin K379 and whole kinesin from bovine brain. The microtubule-activated ATPase is 60 s(-1) at 20 degrees C; Km(Mt) is 5 microM; dissociation constants in the presence of ATP and ADP are 9 microM and 16 microM, respectively. The values of dissociation constants are 5 times larger than for K379. Binding of K332 to microtubules increased the rate of the hydrolysis step from 7 s(-1) to greater than 200 s(-1) and the 2'-(3')-O-(N-methylanthraniloyl) (mant) ADP dissociation step from 0.02 s(-1) to greater than 100 s(-1). At higher ionic strength, more than one ATP is hydrolyzed before dissociation of MtK332 (small processivity). Data are fitted to the kinetic scheme. [equation: see text] Approximate values of rate constants are k1 = 500 s(-1), k2 > or = 200 s(-1), k3k4/(k3 + k4) = 100 s(-1), k(dis) = 80+/-10 s(-1). Two experiments to measure k4 gave 110 s(-1) from the maximum rate of dissociation of mant ADP for reaction of K x ADP with microtubules and 300 s(-1) from extrapolation to zero concentration of rate of binding of mant ADP to MtK. It is proposed that mant ADP dissociation is a two-step process. In the simple scheme, k4 is the effective rate of the two-step release of ADP, k4 = 150 s(-1) to 200 s(-1), and k3 = 150 s(-1) to 200 s(-1) to account for the steady state rate.
对单体人类驱动蛋白构建体K332的动力学机制进行了分析。在没有微管的情况下，ATP酶循环的速率常数与二聚体人类驱动蛋白K379以及牛脑全驱动蛋白非常相似。微管激活的ATP酶在20℃时为60 s⁻¹；Km(Mt)为5 μM；在ATP和ADP存在下的解离常数分别为9 μM和16 μM。解离常数的值比K379大5倍。K332与微管的结合将水解步骤的速率从7 s⁻¹提高到大于200 s⁻¹，并且将2'-(3')-O-(N-甲基邻氨基苯甲酰基)(mant)ADP解离步骤的速率从0.02 s⁻¹提高到大于100 s⁻¹。在较高离子强度下，在MtK332解离之前会水解不止一个ATP（小持续性）。数据拟合到动力学方案中。[方程式：见正文]速率常数的近似值为k1 = 500 s⁻¹，k2≥200 s⁻¹，k3k4/(k3 + k4)=100 s⁻¹，k(dis)=80±10 s⁻¹。测量k4的两个实验得出，对于K x ADP与微管反应，mant ADP最大解离速率对应的k4为110 s⁻¹，从mant ADP与MtK结合速率外推到零浓度得出k4为300 s⁻¹。有人提出mant ADP解离是一个两步过程。在简单方案中，k4是ADP两步释放的有效速率，k4 = 150 s⁻¹至200 s⁻¹，k3 = 150 s⁻¹至200 s⁻¹以解释稳态速率。
