The characterization of myosin-product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction.
The characterization of myosin-product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction.
复制标题
镁离子依赖性腺苷三磷酸酶反应过程中肌球蛋白产物复合物和产物释放步骤的表征。
作者：
Clive R. Bagshaw;D. Trentham
有证据表明，肌球蛋白亚片段-1-ADP复合物，由Mg（2+）和ADP添加到亚片段1，是肌球蛋白Mg（2+）依赖的腺苷三磷酸酶（ATP酶）周转周期内的中间体。通过荧光测量证明了该物质在pH 8和5 ℃下作为稳态中间体的存在，但其浓度变得太低而无法在21 ℃下测量。这是因为控制ADP从亚片段1解离的过程的速率明显依赖于温度（速率在21 ℃为1.4s（-1），在5 ℃为0.07s（-1））。在ATP酶途径中，该反应与相对温度不敏感的过程串联，即亚片段-1-产物复合物的异构化（速率=0.055s（-1）在21 ℃，0.036s（-1）在5 ℃）。通过研究P（i）对核苷酸结合速率的抑制作用，确定了一种肌球蛋白亚片段1-P（i）复合物，其解离平衡常数为1.5mm。P（i）与肌球蛋白亚片段1-ADP复合物的结合似乎较弱。研究表明，P（i）以大于40 s（-1）的速率从亚片段1解离，并证实了在Mg（2+）依赖的ATP酶的基本过程中，在产物释放之前存在肌球蛋白产物异构化。在这种ATP酶机制中，Mg（2+）以复合物的形式与ATP结合，并以复合物的形式与ADP释放。在pH 8的0. 1 m-KCl中，每mol亚片段1释放1.0molH（+），同时伴随着肌球蛋白产物的异构化或P（i）的解离;当ATP与蛋白质结合时，每mol亚片段释放0.23molH（+），而当ADP解离时，每mol亚片段又吸收0.23molH（+）。在实验灵敏度范围内，在涉及ATP裂解的步骤中，没有H（+）释放到培养基中。
Evidence is presented that the myosin subfragment-1-ADP complex, generated by the addition of Mg(2+) and ADP to subfragment 1, is an intermediate within the myosin Mg(2+)-dependent adenosine triphosphatase (ATPase) turnover cycle. The existence of this species as a steady-state intermediate at pH8 and 5 degrees C is demonstrated by fluorescence measurements, but its concentration becomes too low to measure at 21 degrees C. This arises because there is a marked temperature-dependence on the rate of the process controlling ADP dissociation from subfragment 1 (rate=1.4s(-1) at 21 degrees C, 0.07s(-1) at 5 degrees C). In the ATPase pathway this reaction is in series with a relatively temperature-insensitive process, namely an isomerization of the subfragment-1-product complex (rate=0.055s(-1) at 21 degrees C, 0.036s(-1) at 5 degrees C). By means of studies on the P(i) inhibition of nucleotide-association rates, a myosin subfragment-1-P(i) complex was characterized with a dissociation equilibrium constant of 1.5mm. P(i) appears to bind more weakly to the myosin subfragment-1-ADP complex. The studies indicate that P(i) dissociates from subfragment 1 at a rate greater than 40s(-1), and substantiates the existence of a myosin-product isomerization before product release in the elementary processes of the Mg(2+)-dependent ATPase. In this ATPase mechanism Mg(2+) associates as a complex with ATP and is released as a complex with ADP. In 0.1m-KCl at pH8 1.0mol of H(+) is released/mol of subfragment 1 concomitant with the myosin-product isomerization or P(i) dissociation, and 0.23 mol of H(+) is released/mol of subfragment when ATP binds to the protein, but 0.23 mol of H(+) is taken up again from the medium when ADP dissociates. Within experimental sensitivity no H(+) is released into the medium in the step involving ATP cleavage.