Mechanism of actomyosin adenosine triphosphatase. Evidence that adenosine 5'-triphosphate hydrolysis can occur without dissociation of the actomyosin complex.
Mechanism of actomyosin adenosine triphosphatase. Evidence that adenosine 5'-triphosphate hydrolysis can occur without dissociation of the actomyosin complex.
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肌动球蛋白三磷酸腺苷酶的机制。
作者：
L. Stein;R. P. Schwarz;P. Chock;E. Eisenberg
我们研究了肌球蛋白ATPase循环中决定最大ATPase速率(Vmax)的步骤以及当ATPase活性接近Vmax时发生的肌球蛋白亚片段1(S-1)与肌动蛋白的结合。我们发现，肌动蛋白S-1 ATPase的最大周转速率比肌动蛋白ATPase的最大周转速率快5倍。因此，周期中的另一步必须比初始PI突发的前向速率慢得多。如果这一较慢的步骤只发生在S-1与肌动蛋白结合时，如最初由Lymn-Taylor模型预测的那样，则稳态时ATP酶活性和S-1与肌动蛋白结合的比例应该几乎是随着肌动蛋白浓度的增加而平行增加的。根据停流仪测定的浊度，S-1与肌动蛋白结合的比例与肌动蛋白ATPase活性一样，与肌动蛋白浓度呈双曲线关系，渐近接近100%。然而，使50%的S-1与肌动蛋白结合所需的肌动蛋白浓度大约是达到一半最大ATPase活性所需的肌动蛋白浓度的4倍。因此，正如以前在0摄氏度发现的那样，在15摄氏度时，当ATPase接近Vmax时，S-1的大部分与肌动蛋白解离，这表明在最初的PI猝发之后的缓慢的一级转变(从难治状态到非难治状态的转变)肯定是ATPase循环中最慢的步骤。停流研究还表明，无论混合顺序如何，S-1、肌动蛋白和三磷酸腺苷几乎在瞬间达到稳态浊度水平。因此，在稳态下观察到的S-1与肌动蛋白的结合是由于S-1-三磷酸腺苷与Acto-S-1-三磷酸腺苷之间的快速平衡，并在较高的肌动蛋白浓度下向Acto-S-1-三磷酸腺苷转移。此外，S-1-三磷酸腺苷和S-1-ADP.PI(在初始PI爆发后立即出现的状态)与肌动蛋白似乎具有相同的结合常数。因此，在较高的肌动蛋白浓度下，S-1-三磷酸腺苷和S-1-ADP.PI都与各自的肌动蛋白复合体快速平衡。虽然在很高的肌动蛋白浓度下，S-1-三磷酸腺苷和S-1-ADP.PI几乎完全与肌动蛋白结合，但在很高的肌动蛋白浓度下，ATPase活性并没有受到抑制。这有力地表明，无论S-1是与肌动蛋白结合还是与肌动蛋白解离，最初的PI猝发和随后的缓慢限速转变(从难治状态到非难治状态的转变)都以大致相同的速度发生。因此，我们得出结论，S-1不必在每一次三磷酸腺苷分子被水解时都与肌动蛋白解离。
We have investigated the steps in the actomyosin ATPase cycle that determine the maximum ATPase rate (Vmax) and the binding between myosin subfragment one (S-1) and actin which occurs when the ATPase activity is close to Vmax. We find that the forward rate constant of the initial ATP hydrolysis (initial Pi burst) is about 5 times faster than the maximum turnover rate of the actin S-1 ATPase. Thus, another step in the cycle must be considerably slower than the forward rate of the initial Pi burst. If this slower step occurs only when S-1 is complexed with actin, as originally predicted by the Lymn-Taylor model, the ATPase activity and the fraction of S-1 bound to actin in the steady state should increase almost in parallel as the actin concentration is increased. As measured by turbidity determined in the stopped-flow apparatus, the fraction of S-1 bound to actin, like the ATPase activity, shows a hyperbolic dependence on actin concentration, approaching 100% asymptotically. However, the actin concentration required so that 50% of the S-1 is bound to actin is about 4 times greater than the actin concentration required for half-maximal ATPase activity. Thus, as previously found at 0 degrees C, at 15 degrees C much of the S-1 is dissociated from actin when the ATPase is close to Vmax, showing that a slow first-order transition which follows the initial Pi burst (the transition from the refractory to the nonrefractory state) must be the slowest step in the ATPase cycle. Stopped-flow studies also reveal that the steady-state turbidity level is reached almost instantaneously after the S-1, actin, and ATP are mixed, regardless of the order of mixing. Thus, the binding between S-1 and actin which is observed in the steady state is due to a rapid equilibrium between S-1--ATP and acto--S-1--ATP which is shifted toward acto-S-1--ATP at high actin concentration. Furthermore, both S-1--ATP and S-1--ADP.Pi (the state occurring immediately after the initial Pi burst) appear to have the same binding constant to actin. Thus, at high actin concentration both S-1--ATP and S-1--ADP.Pi are in rapid equilibrium with their respective actin complexes. Although at very high actin concentration almost complete binding of S-1--ATP and S-1--ADP.Pi to actin occurs, there is no inhibition of the ATPase activity at high actin concentration. This strongly suggests that both the initial Pi burst and the slow rate-limiting transition which follows (the transition from the refractory to the nonrefractory state) occur at about the same rates whether the S-1 is bound to or dissociated from actin. We, therefore, conclude that S-1 does not have to dissociate from actin each time an ATP molecule is hydrolyzed.