Ma Y Z, Taylor E W
Department of Molecular Genetics and Cell Biology, University of Chicago, Cummings Life Science Center, Illinois 60637, USA.
Biochemistry. 1995 Oct 10;34(40):13242-51. doi: 10.1021/bi00040a040.
A six-step mechanism is derived for the activation of kinesin K379 ATPase by microtubules. The data are fitted by the kinetic scheme [Formula see text] where T, D, and P refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively; MtK refers to the complex of a K379 unit with the microtubule binding site. The initial binding and release steps, 1 and 6, are treated as rapid equilibria: k2 = 200 s-1, k3 = 100 s-1, k5 = 35-40 s-1, maximum steady-state rate = 25 s-1 (50 mM NaCl, 20 degrees C). k2 was obtained from the maximum rate of fluorescence enhancement with mant-ATP as substrate, k3 was obtained from the hydrolysis transient phase for ATP or mant-ATP, and k5 was obtained from the rate of decrease in fluorescence of mant-ADP in the reaction [Formula see text]. A large excess of ATP was present with the Mt to block rebinding of mant-ADP. The rate was measured as a function of microtubule concentration and extrapolated to give the maximum rate k5. The same method was used to obtain k5 for ADP by mixing K.ADP with microtubules plus excess mant-ATP. The enhancement of fluorescence for the binding of mant-ATP is followed by a decrease in fluorescence with a rate constant of 35-40 s-1. Since the decrease must occur after hydrolysis, it may be correlated with a step or steps leading to the low fluorescence MtK.D state. In the kinetic scheme, steps 4 and 5 both contribute to determining the maximum turnover rate. At higher ionic strengths or lower protein concentrations, the MtK complex is dissociated by ATP. The maximum rate is 12 +/- 2 s-1 in 50 mM NaCl; consequently, hydrolysis occurs before dissociation. The dissociation constant of MtK in the presence of ADP is twice as large as the dissociation constant in the presence of ATP and four times larger than the KM for microtubule activation. The proposed kinetic scheme, which treats the K379 units of a dimer as independent, provides a satisfactory description of the transient and steady-state properties of the system with the possible exception of results at very low substrate concentrations.
推导了一种微管激活驱动蛋白K379 ATP酶的六步机制。数据通过动力学方案[公式见原文]进行拟合，其中T、D和P分别指三磷酸核苷酸、二磷酸核苷酸和无机磷酸；MtK指K379单元与微管结合位点的复合物。初始结合和释放步骤（步骤1和6）被视为快速平衡：k2 = 200 s-1，k3 = 100 s-1，k5 = 35 - 40 s-1，最大稳态速率 = 25 s-1（50 mM NaCl，20℃）。k2是通过以mant-ATP为底物时荧光增强的最大速率获得的，k3是通过ATP或mant-ATP的水解瞬态阶段获得的，k5是通过反应[公式见原文]中mant-ADP荧光降低的速率获得的。向Mt中加入大量过量的ATP以阻止mant-ADP的重新结合。速率作为微管浓度的函数进行测量，并外推以得到最大速率k5。通过将K.ADP与微管加过量mant-ATP混合，使用相同的方法获得ADP的k5。mant-ATP结合时荧光增强之后是荧光以35 - 40 s-1的速率常数降低。由于这种降低一定发生在水解之后，它可能与导致低荧光MtK.D状态的一个或多个步骤相关。在动力学方案中，步骤4和5都有助于确定最大周转速率。在较高离子强度或较低蛋白质浓度下，MtK复合物会被ATP解离。在50 mM NaCl中最大速率为12±2 s-1；因此，水解发生在解离之前。在存在ADP的情况下，MtK的解离常数是存在ATP时解离常数的两倍，并且比微管激活的KM大四倍。所提出的将二聚体的K379单元视为独立的动力学方案，除了在非常低底物浓度下得到的结果可能例外，对该系统的瞬态和稳态特性提供了令人满意的描述。
