Time-dependent inhibition of recA protein-catalyzed ATP hydrolysis by ATP gamma S: Evidence for a rate-determining isomerization of the recA-ssDNA complex
Time-dependent inhibition of recA protein-catalyzed ATP hydrolysis by ATP gamma S: Evidence for a rate-determining isomerization of the recA-ssDNA complex
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DOI：
10.1021/bi970576
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发表时间：
1997-06-24
期刊：
影响因子：
2.9
通讯作者：
Bryant, FR
中科院分区：
文献类型：
--
作者：
Paulus, BF;Bryant, FR
ATP类似物ATP γ S是recA蛋白催化的ssDNA依赖性ATP水解反应的竞争性抑制剂。然而，ATP γ S的抑制程度以时间依赖性方式变化，并且与两步结合机制一致。在第一步中，ATP γ S在快速平衡步骤（K-D = 50 μ M）中与recA-ssDNA复合物结合。该初始结合步骤之后，recA-ssDNA-ATP γ S复合物异构化为新的构象状态，其中ATP γ S以显著更高的亲和力结合（总K-D = 0.3 μ M）。该异构化之后，ATP γ S缓慢水解为ADP和硫代磷酸（0.01 min（-1））。ATP γ S介导的异构化步骤的一级速率常数（20 min（-1）），尽管显著大于ATP γ S水解的速率，但与recA蛋白催化的ATP水解反应的稳态速率常数相同，这些结果与动力学模型一致，其中recA-ssDNA复合物的ATP介导的异构化代表了recA蛋白催化的ssDNA依赖性ATP水解反应途径的决定步骤。
The ATP analog ATP gamma S is a competitive inhibitor of the recA protein-catalyzed ssDNA-dependent ATP hydrolysis reaction. The degree of inhibition by ATP gamma S, however, changes in a time-dependent manner and is consistent with a two step binding mechanism. In the first step, ATP gamma S binds to the recA-ssDNA complex in a rapid equilibrium step (K-D = 50 mu M). This initial binding step is followed by an isomerization of the recA-ssDNA-ATP gamma S complex to a new conformational state in which ATP gamma S is bound with a significantly higher affinity (overall K-D = 0.3 mu M) This isomerization is followed by the slow hydrolysis of ATP gamma S to ADP and thiophosphate (0.01 min(-1)). The first-order rate constant for the ATP gamma S-mediated isomerization step (20 min(-1)), although significantly greater than the rate of ATP gamma S hydrolysis, is identical to the steady-state rate constant for the recA protein-catalyzed ATP hydrolysis reaction, These results are consistent with a kinetic model in which an ATP-mediated isomerization of the recA-ssDNA complex represents the rate-determining step on the recA protein-catalyzed ssDNA-dependent ATP hydrolysis reaction pathway.