p21N-ras对GTP水解的动力学分析。基础GTP酶机制。

Kinetic analysis of the hydrolysis of GTP by p21N-ras. The basal GTPase mechanism.

作者信息

Neal S E, Eccleston J F, Hall A, Webb M R

机构信息

Division of Physical Biochemistry, National Institute for Medical Research, London, United Kingdom.

出版信息

J Biol Chem. 1988 Dec 25;263(36):19718-22.

PMID:2848838

Abstract

The rate constants have been determined for elementary steps in the basal GTPase mechanism of normal p21N-ras (Gly-12) and an oncogenic mutant (Asp-12): namely GTP binding, hydrolysis, phosphate release, and GDP release. By extrapolation from data at lower temperatures, the GTP association rate constant at 37 degrees C is 1.4 x 10(8) M-1 s-1 for the normal protein and 4.8 x 10(8) M-1 s-1 for the mutant. Other rate constants were measured directly at 37 degrees C, and three processes have similar slow values. GTP dissociation is at 1.0 x 10(-4) s-1 (normal) and 5.0 x 10(-4) s-1 (mutant). The hydrolysis step is at 3.4 x 10(-4) s-1 (normal) and 1.5 x 10(-4) s-1 (mutant). GDP dissociates at 4.2 x 10(-4) s-1 (normal) and 2.0 x 10(-4) s-1 (mutant). GDP association rate constants are similar to those for GTP, 0.5 x 10(8) M-1 s-1 for normal and 0.7 x 10(8) M-1 s-1 for mutant. Both hydrolysis and GDP release therefore contribute to rate limitation of the basal GTPase activity. There are distinct differences (up to 5-fold) between rate constants for the normal and mutant proteins at a number of steps. The values are consistent with the reduced GTPase activity for this mutant and suggest little difference between normal and mutant proteins in the relative steady-state concentrations of GTP and GDP complexes that may represent active and inactive states. The results are discussed in terms of the likely role of p21ras in transmembrane signalling.

摘要

已测定正常p21N-ras（甘氨酸-12）和致癌突变体（天冬氨酸-12）的基础GTP酶机制中各个基本步骤的速率常数，即GTP结合、水解、磷酸释放和GDP释放。通过从较低温度下的数据外推，正常蛋白在37℃时的GTP缔合速率常数为1.4×10⁸ M⁻¹ s⁻¹，突变体为4.8×10⁸ M⁻¹ s⁻¹。其他速率常数在37℃时直接测量，三个过程具有相似的缓慢值。GTP解离速率为1.0×10⁻⁴ s⁻¹（正常）和5.0×10⁻⁴ s⁻¹（突变体）。水解步骤速率为3.4×10⁻⁴ s⁻¹（正常）和1.5×10⁻⁴ s⁻¹（突变体）。GDP解离速率为4.2×10⁻⁴ s⁻¹（正常）和2.0×10⁻⁴ s⁻¹（突变体）。GDP缔合速率常数与GTP的相似，正常为0.5×10⁸ M⁻¹ s⁻¹，突变体为0.7×10⁸ M⁻¹ s⁻¹。因此，水解和GDP释放均导致基础GTP酶活性的速率限制。在多个步骤中，正常蛋白和突变蛋白的速率常数存在明显差异（高达5倍）。这些值与该突变体降低的GTP酶活性一致，并表明正常蛋白和突变蛋白在可能代表活性和非活性状态的GTP和GDP复合物的相对稳态浓度方面差异不大。根据p21ras在跨膜信号传导中的可能作用对结果进行了讨论。