Wolcott R G, Boyer P D
J Supramol Struct. 1975;3(2):154-61. doi: 10.1002/jss.400030208.
A new approach to the direct estimation of the value of the off constant for dissociation of ATP from myosin subfragment 1 (S1) has been developed. From measurements of the extremely slow rate of release of [32P] - ATP formed from 32P(i) by S1 catalysis and the amount of rapidly formed [32P] - ATP tightly bound to S1, the value of the off constant is approximately 2.8 X 10(-4) sec -1 at pH 7.4. The concentration dependencies for P(i) in equilibrium H18 OH exchange and for (32)P(j) incorporation into myosin-bound ATP give direct measurements of the dissociation constant of P(i) from S1. Both approaches show that the enzyme has a very low affinity for P(i), with an apparent K(d) of greater than 400 mM. Measurement of the average number of water oxygens incorporated into P(i) released from ATP by S1-catalyzed hydrolysis in the presence of Mg2+ suggests that the hydrolytic step reverses an average of at least 5.5 times for each ATP cleaved. With the Ca2+ -activated hydrolysis, less than one oxygen from water appears in each P(i) released. This finding is indicative of a possible isotope effect in the attack of water on the terminal phosphoryl group of ATP.
已开发出一种直接估算ATP从肌球蛋白亚片段1（S1）解离的解离常数（off常数）值的新方法。通过测量由S1催化从32P(i)形成的[32P]-ATP的极缓慢释放速率以及紧密结合到S1上的快速形成的[32P]-ATP的量，在pH 7.4时，off常数的值约为2.8×10(-4)秒-1。平衡H18OH交换中P(i)的浓度依赖性以及(32)P(j)掺入与肌球蛋白结合的ATP中的浓度依赖性，可直接测量P(i)从S1的解离常数。两种方法均表明该酶对P(i)的亲和力非常低，表观K(d)大于400 mM。在Mg2+存在下，通过S1催化水解从ATP释放的P(i)中掺入的水氧原子平均数的测量表明，每裂解一个ATP，水解步骤平均至少逆转5.5次。在Ca2+激活的水解过程中，每个释放的P(i)中来自水的氧原子少于一个。这一发现表明水对ATP末端磷酰基的攻击可能存在同位素效应。
