MECHANISM OF MICROTUBULE KINESIN ATPASE
MECHANISM OF MICROTUBULE KINESIN ATPASE
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DOI：
10.1021/bi00040a040
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发表时间：
1995-10-10
期刊：
影响因子：
2.9
通讯作者：
TAYLOR, EW
中科院分区：
文献类型：
--
作者：
MA, YZ;TAYLOR, EW
提出了微管激活驱动蛋白K379 ATP酶的六步机制。通过动力学方案[GRAPHICS]拟合数据，其中T、D和P分别指核苷酸三磷酸、核苷酸二磷酸和无机磷酸; 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1和6被视为快速平衡; k（2）= 200 s（-1），k（3）= 100 s（-1），k（5）= 35-40 s（-1），最大稳态速率= 25 s（-1）（50 mM NaCl，20 ℃）。k（2）由以mant-ATP为底物的荧光增强的最大速率获得，k（3）由ATP或mant-ATP的水解过渡相获得，k（5）由反应K中mant-ADP的荧光降低速率获得。D + Mt可逆箭头MtK。D可逆箭头MtK + D。大量过量的ATP与MT一起存在，以阻止mant-ADP的再结合。速率作为微管浓度的函数进行测量，并外推得到最大速率k（5）。使用相同的方法通过混合K来获得ADP的k（5）。ADP和微管加上过量的mant-ATP。mant-ATP结合的荧光增强后，荧光减弱，速率常数为35-40 s（-1）。由于降低必须在水解后发生，因此它可能与导致低荧光MtK的一个或多个步骤相关。D州。在动力学方案中，步骤4和5都有助于确定最大周转率。在较高的离子强度或较低的蛋白质浓度下，MtK复合物被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ADP存在下的MtK的解离常数是ATP存在下的解离常数的两倍，并且是微管活化的K-M的四倍。建议的动力学方案，它把K379单位的二聚体作为独立的，提供了一个令人满意的描述的瞬态和稳态性能的系统与可能的例外，在非常低的底物浓度的结果。
A six-step mechanism is derived for the activation of kinesin K379 ATPase by microtubules. The data are fitted by the kinetic scheme[GRAPHICS]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; k(2) = 200 s(-1), k(3) = 100 s(-1), k(5) = 35-40 s(-1), maximum steady-state rate = 25 s(-1) (50 mM NaCl, 20 degrees C). k(2) was obtained from the maximum rate of fluorescence enhancement with mant-ATP as substrate, k(3) was obtained from the hydrolysis transient phase for ATP or mant-ATP, and k(5) was obtained from the rate of decrease in fluorescence of mant-ADP in the reaction K . D + Mt reversible arrow MtK . D reversible arrow MtK + D. 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 k(5). The same method was used to obtain k(5) for ADP by mixing K . ADP with microtubules plus excess mant-ATP. The enhancement of fluorescence for th 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 K-M 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.