Srinivasa S P, Watson N, Overton M C, Blumer K J
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1998 Jan 16;273(3):1529-33. doi: 10.1074/jbc.273.3.1529.
GTP hydrolysis by guanine nucleotide-binding proteins, an essential step in many biological processes, is stimulated by GTPase-activating proteins (GAPs). The mechanisms whereby GAPs stimulate GTP hydrolysis are unknown. We have used mutational, biochemical, and structural data to investigate how RGS4, a GAP for heterotrimeric G protein alpha subunits, stimulates GTP hydrolysis. Many of the residues of RGS4 that interact with Gi alpha 1 are important for GAP activity. Furthermore, optimal GAP activity appears to require the additive effects of interactions along the RGS4-G alpha interface. GAP-defective RGS4 mutants invariably were defective in binding G alpha subunits in their transition state; furthermore, the apparent strengths of GAP and binding defects were correlated. Thus, none of these residues of RGS4, including asparagine 128, the only residue positioned at the active site of Gi alpha 1, is required exclusively for catalyzing GTP hydrolysis. These results and structural data (Tesmer, J. G. G., Berman, D. M., Gilman, A. G., and Sprang, S. R. (1997) Cell 89, 251-261) indicate that RGS4 stimulates GTP hydrolysis primarily by stabilizing the transition state conformation of the switch regions of the G protein, favoring the transition state of the reactants. Therefore, although monomeric and heterotrimeric G proteins are related, their GAPs have evolved distinct mechanisms of action.
鸟嘌呤核苷酸结合蛋白催化的GTP水解是许多生物过程中的关键步骤，这一过程受GTP酶激活蛋白（GAP）的刺激。然而，GAP刺激GTP水解的具体机制尚不清楚。我们通过突变、生化和结构数据研究了异三聚体G蛋白α亚基的GAP——RGS4如何刺激GTP水解。RGS4中许多与Giα1相互作用的残基对GAP活性至关重要。此外，最佳的GAP活性似乎需要沿RGS4 - Gα界面相互作用的累加效应。GAP缺陷型RGS4突变体在结合处于过渡态的Gα亚基时总是存在缺陷；此外，GAP缺陷和结合缺陷的表观强度呈正相关。因此，RGS4的这些残基，包括位于Giα1活性位点的唯一残基天冬酰胺128，都不是催化GTP水解所必需的。这些结果和结构数据（Tesmer, J. G. G., Berman, D. M., Gilman, A. G., and Sprang, S. R. (1997) Cell 89, 251 - 261）表明，RGS4主要通过稳定G蛋白开关区域的过渡态构象来刺激GTP水解，从而有利于反应物的过渡态。因此，尽管单体G蛋白和异三聚体G蛋白相关，但它们的GAP进化出了不同的作用机制。
