Zor T, Bar-Yaacov M, Elgavish S, Shaanan B, Selinger Z
Department of Biological Chemistry, the Kuhne Minerva Center for studies of visual transduction, The Hebrew University of Jerusalem, Israel.
Eur J Biochem. 1997 Oct 1;249(1):330-6. doi: 10.1111/j.1432-1033.1997.00330.x.
Signaling by guanine-nucleotide-binding proteins (G-proteins) occurs when they are charged with GTP, while hydrolysis of the bound nucleotide turns the signaling off. Despite a wealth of biochemical and structural information, the mechanism of GTP hydrolysis by G-proteins remains controversial. We have employed substrate-assisted catalysis as a novel approach to study catalysis by G-proteins. In these studies, we have used diaminobenzophenone-phosphonoamidate-GTP, a unique GTP analog bearing the functional groups that are missing in the GTPase-deficient [Leu227]G(s alpha) mutant. This mutant, found in various human tumors, fails to hydrolyze GTP for an extended period. In contrast, the GTP analog is hydrolyzed by this mutant and by the wild-type enzyme at the same rate. On the other hand, modification of G(s alpha) by cholera toxin, which catalyses ADP-ribosylation of Arg201 of G(s alpha), decreased the rates of hydrolysis of both GTP and its analog by 95%. These results attest to the specificity of the GTP analog as a unique substrate for the [Leu227]G(s alpha) mutant and to the essential role of Gln227 in GTP hydrolysis. Furthermore, the finding that the GTP analog was hydrolyzed at the same rate as GTP by the wild-type enzyme, favors a model in which formation of a pentavalent transition state intermediate, presumably stabilized by the catalytic glutamine, is not the rate-limiting step of the GTPase reaction.
鸟嘌呤核苷酸结合蛋白（G蛋白）在结合GTP时发生信号传导，而结合核苷酸的水解则会关闭信号传导。尽管有大量的生化和结构信息，但G蛋白水解GTP的机制仍存在争议。我们采用底物辅助催化作为研究G蛋白催化作用的新方法。在这些研究中，我们使用了二氨基二苯甲酮-膦酰胺基-GTP，这是一种独特的GTP类似物，带有GTP酶缺陷型[Leu227]G（sα）突变体中缺失的官能团。在各种人类肿瘤中发现的这种突变体，在很长一段时间内都无法水解GTP。相比之下，这种GTP类似物被该突变体和野生型酶以相同的速率水解。另一方面，霍乱毒素对G（sα）的修饰，催化G（sα）的Arg201的ADP核糖基化，使GTP及其类似物的水解速率降低了95%。这些结果证明了GTP类似物作为[Leu227]G（sα）突变体的独特底物的特异性，以及Gln227在GTP水解中的重要作用。此外，野生型酶对GTP类似物的水解速率与GTP相同这一发现支持了一种模型，即在该模型中，由催化性谷氨酰胺稳定的五价过渡态中间体的形成不是GTP酶反应的限速步骤。
