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):13233-41. doi: 10.1021/bi00040a039.
The kinetic mechanism of the human kinesin ATPase motor domain K379, expressed in Escherichia coli, was determined by transient and steady-state kinetic studies. The steps in nucleotide binding were measured using the fluorescent substrate analogues, methylanthraniloyl ATP (mant-ATP) and mant-ADP. Both nucleotides gave a two-step fluorescence signal, an increase followed by a decrease, which indicates that two isomerizations are induced by nucleotide binding. The ATPase mechanism is fitted by a six-step reaction: [formula: see text] where, T, D, and P refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively; K(T) and K(D) are states in rapid equilibrium with the free nucleotide. A set of kinetic constants for 20 degrees C 50 mM NaCl is K1 = 2 x 10(4) M-1, k2 = 200 s-1, k3 = 9 s-1, k5 = 0.01 s-1, and K6 = 2 x 10(-5) M. Values of K1 and K6 are estimates for mant-ATP and mant-ADP, respectively. ADP dissociation is the rate-limiting step. The rate constant for a decrease in fluorescence for the transitions from the high fluorescence K.T state to the low fluorescence K.D state is equal to k3, the rate constant of the hydrolysis step measured by quench flow experiments. The decrease could occur in step 3 or step 4 if k4 > k3.(ABSTRACT TRUNCATED AT 250 WORDS)
通过瞬态和稳态动力学研究确定了在大肠杆菌中表达的人驱动蛋白ATP酶运动结构域K379的动力学机制。使用荧光底物类似物甲基蒽酰胺ATP（mant-ATP）和mant-ADP测量核苷酸结合的步骤。两种核苷酸都给出了两步荧光信号，先增加后降低，这表明核苷酸结合诱导了两种异构化。ATP酶机制由六步反应拟合：[公式：见正文]，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；K（T）和K（D）是与游离核苷酸处于快速平衡的状态。20℃、50mM NaCl条件下的一组动力学常数为K1 = 2×10⁴ M⁻¹，k2 = 200 s⁻¹，k3 = 9 s⁻¹，k5 = 0.01 s⁻¹，K6 = 2×10⁻⁵ M。K1和K6的值分别是mant-ATP和mant-ADP的估计值。ADP解离是限速步骤。从高荧光K.T状态转变为低荧光K.D状态时荧光降低的速率常数等于k3，即通过淬灭流动实验测量的水解步骤的速率常数。如果k4 > k3，降低可能发生在步骤3或步骤4中。（摘要截断于250字）
