在大肠杆菌中表达的果蝇驱动蛋白最小运动结构域。纯化及动力学特性分析。

Drosophila kinesin minimal motor domain expressed in Escherichia coli. Purification and kinetic characterization.

作者信息

Huang T G, Hackney D D

机构信息

Department of Biological Sciences Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.

出版信息

J Biol Chem. 1994 Jun 10;269(23):16493-501.

PMID:8206959

Abstract

A truncated motor domain of the alpha subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP. This domain (designated DKH340) extends from the N terminus to amino acid 340. The isolated protein contains a stoichiometric level of tightly bound ADP and has a low basal rate of ATP hydrolysis of 0.029 +/- 0.002 s-1 in the absence of microtubules. The rate of release of bound ADP is 0.026 +/- 0.003 s-1. The approximate equality of the ADP release rate and the steady state ATPase rate indicates that ADP release is the rate-limiting step in ATP hydrolysis in the absence of microtubules. The rate of ATP hydrolysis is stimulated 3000 fold-by addition of microtubules (MT) (kcat = 80 s-1; KMT0.5,ATPase = 160 nM for half-saturation of the ATPase rate by microtubules at saturating ATP levels; KMT0.5ATPase = 43 microns for half-saturation of the ATPase rate by ATP at saturating microtubule levels). Binding of DKH340 to MTs is biphasic in the presence of adenosine 5-(beta-gamma-imido)t-riphosphate. One DKH340 binds tightly per tubulin heterodimer, but greater than one DKH340/tubulin heterodimer can be bound at higher ratios of DKH340/microtubules. In the presence of MgATP, KMT0.5,Binding for physical binding of DKH340 to microtubules is weaker than KMT0.5,ATPase for stimulation of hydrolysis. These results are consistent with a model in which DKH340 cycles on and off the microtubule during hydrolysis of each ATP molecule. For this model, the kcat/KMT0.5,ATPase ratio of 5 x 10(8) M-1 s-1 is at least as large as the bimolecular rate constant for association with microtubules, and this value approaches the diffusion controlled limit. Nucleotide-free DKH340 can be produced by gel filtration in the absence of Mg2+, but it reforms tightly bound ADP slowly in the presence of MgATP (t1/2 > or = 10 min), and thus it is likely to be in a conformational state which is not produced during steady state ATP hydrolysis.

摘要

通过在大肠杆菌中表达获得了果蝇驱动蛋白α亚基的截短运动结构域，并在MgATP存在下纯化至同质。该结构域（命名为DKH340）从N端延伸至氨基酸340。分离出的蛋白质含有化学计量水平的紧密结合的ADP，在无微管存在时，其基础ATP水解速率较低，为0.029±0.002 s⁻¹。结合的ADP释放速率为0.026±0.003 s⁻¹。ADP释放速率与稳态ATP酶速率大致相等，表明在无微管存在时，ADP释放是ATP水解的限速步骤。添加微管（MT）可使ATP水解速率提高3000倍（kcat = 80 s⁻¹；在饱和ATP水平下，微管使ATP酶速率达到半饱和时的KMT0.5,ATPase = 160 nM；在饱和微管水平下，ATP使ATP酶速率达到半饱和时的KMT0.5ATPase = 43微米）。在腺苷5-(β-γ-亚氨基)三磷酸存在下，DKH340与MTs的结合是双相的。每个微管蛋白异二聚体紧密结合一个DKH340，但在DKH340/微管的比例更高时，每个微管蛋白异二聚体可结合超过一个DKH340。在MgATP存在下，DKH340与微管物理结合的KMT0.5,Binding比微管刺激水解的KMT0.5,ATPase弱。这些结果与一个模型一致，即在每个ATP分子水解过程中，DKH340在微管上循环结合和解离。对于该模型，kcat/KMT0.5,ATPase比值为5×10⁸ M⁻¹ s⁻¹，至少与与微管结合的双分子速率常数一样大，且该值接近扩散控制极限。在无Mg²⁺的情况下，通过凝胶过滤可产生无核苷酸的DKH340，但在MgATP存在下，它会缓慢重新形成紧密结合的ADP（t1/2≥10分钟），因此它可能处于稳态ATP水解过程中未产生的构象状态。