Individual rate constants for the interaction of Ras proteins with GTPase-activating proteins determined by fluorescence spectroscopy
Individual rate constants for the interaction of Ras proteins with GTPase-activating proteins determined by fluorescence spectroscopy
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DOI：
10.1021/bi962556y
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发表时间：
1997-04-15
期刊：
影响因子：
2.9
通讯作者：
Wittinghofer, A
中科院分区：
文献类型：
--
作者：
Ahmadian, MR;Hoffmann, U;Wittinghofer, A
用PAS蛋白活性部位鸟嘌呤核苷酸的荧光衍生物测定了H-、K-和N-RAS与GAP-334和NF1-333相互作用的速率常数。NF1-333的停流实验表明，与结合反应对应的快速浓度依赖的初始相对应于结合反应，然后是对应于水解反应的较慢相。当PAS与非水解性类似物MANT-GppNHp结合时，只观察到浓度依赖的第一相。用Ras mant-GppNHp NF1-333络合物测定了PAS-GaP络合物的离解速率常数。用GAP-334作催化剂，第一相的浓度依赖性太快，不能用停流法测量，但随后的化学裂解反应发生的速度与用NF1-333时相似(5-10 S(-1))。对于GAP-334和NF1-333，在快速达到初始平衡后，与RAS(.)mant-GppNHp混合间隙没有进一步的时间依赖变化。所获得的结果为了解GAP催化的GTP酶在PAS上反应的各个步骤提供了新的见解。它们不需要假设在GTP水解之前发生限速步骤。
Individual rate constants for the interaction of H-, K-, and N-Ras with GAP-334 and NF1-333 were determined using fluorescent derivatives of guanine nucleotides at the active site of the Pas proteins. Stopped-flow experiments with NF1-333 show a fast concentration-dependent initial phase corresponding to the binding reaction followed by a slower phase, which corresponds to the hydrolysis reaction. With Pas bound to the nonhydrolyzable analogue mant-GppNHp, only the concentration-dependent first phase was observed. The Ras mant-GppNHp NF1-333 complexes were also used to measure dissociation rate constants of the Pas-GAP complexes. Using GAP-334 as the catalyst, the concentration-dependent first phase was too fast to be measured by the stopped-flow method, but the subsequent chemical cleavage reaction occurred at a similar rate (5-10 s(-1)) to that seen with NF1-333. With both GAP-334 and NF1-333, after rapidly reaching the initial equilibrium, there was no further time-dependent change on mixing GAPs with Ras(.)mant-GppNHp. The results obtained provide new insights into the individual steps of the GAP-catalyzed GTPase reaction on Pas. They do not require the postulation of a rate-limiting step occurring before GTP hydrolysis.