Chakrabarti Partha P, Daumke Oliver, Suveyzdis Yan, Kötting Carsten, Gerwert Klaus, Wittinghofer Alfred
Abteilung Strukturelle Biologie, Max-Planck-Institut für molekulare Physiologie, D-44227 Dortmund, Germany.
J Mol Biol. 2007 Apr 6;367(4):983-95. doi: 10.1016/j.jmb.2006.11.022. Epub 2006 Nov 10.
Rap1 and Rap2 are the only small guanine nucleotide-binding proteins of the Ras superfamily that do not use glutamine for GTP hydrolysis. Moreover, Rap1GAP, which stimulates the GTPase reaction of Rap1 10(5)-fold, does not have the classical "arginine finger" like RasGAP but presumably, introduces an asparagine residue into the active site. Here, we address the requirements of this unique reaction in detail by combining various biochemical methods, such as fluorescence spectroscopy, stopped-flow and time-resolved Fourier transform infrared spectroscopy (FTIR). The fluorescence spectroscopic assay monitors primarily protein-protein interaction steps, while FTIR resolves simultaneously the elementary steps of functional groups labor-free, but it is less sensitive and needs higher concentrations. Combining both methods allows us to distinguish weather mechanistic defects caused by mutation are due to affinity or due to functionality. We show that several mutations of Asn290 block catalysis. Some of the mutants, however, still form a complex with Rap1*GDP in the presence of BeF(x) but not AlF(x), supporting the notion that fluoride complexes are indicators of the ground versus transition state. Mutational analysis also shows that Thr61 is not required for catalysis. While replacement of Thr61 of Rap1 by Leu eliminates GTPase activation by Rap1GAP, the T61A and T61Q mutants have only a minor effect on catalysis, but change the relative rates of cleavage and (P(i)(-)) release. While Rap1GAP(N290A) is completely inactive on wild-type Rap1, it can act on Rap1(T61Q), arguing that Asn290 in trans has a role in catalysis similar to that of the intrinsic Gln in Ras and Rho. Finally, since FTIR works at high, and thus mostly saturating, concentrations, it can clearly separate effects on affinity from purely catalytic modifications, showing that Arg388, conserved between RapGAPs and mutated in the homologous RheBGAP Tuberin, affects binding affinity severely but has no effect on the cleavage reaction itself.
Rap1和Rap2是Ras超家族中仅有的两种不利用谷氨酰胺进行GTP水解的小GTP结合蛋白。此外，能将Rap1的GTP酶反应速率提高10⁵倍的Rap1GAP，不像RasGAP那样具有经典的“精氨酸指”，但推测它会将一个天冬酰胺残基引入活性位点。在此，我们通过结合多种生化方法，如荧光光谱法、停流法和时间分辨傅里叶变换红外光谱法（FTIR），详细研究了这一独特反应的要求。荧光光谱分析主要监测蛋白质-蛋白质相互作用步骤，而FTIR能同时无损伤地解析官能团的基本步骤，但灵敏度较低且需要更高浓度。将这两种方法结合起来使我们能够区分由突变引起的机制缺陷是由于亲和力还是由于功能。我们表明，天冬酰胺290的几个突变会阻断催化作用。然而，一些突变体在存在BeF(x)而非AlF(x)的情况下仍能与Rap1*GDP形成复合物，这支持了氟化物复合物是基态与过渡态指标的观点。突变分析还表明，催化作用不需要苏氨酸61。虽然将Rap1的苏氨酸61替换为亮氨酸会消除Rap1GAP对GTP酶的激活作用，但T61A和T61Q突变体对催化作用只有轻微影响，但会改变裂解和（Pi⁻）释放的相对速率。虽然Rap1GAP(N290A)对野生型Rap1完全无活性，但它能作用于Rap1(T61Q)，这表明反式的天冬酰胺290在催化中所起的作用类似于Ras和Rho中固有的谷氨酰胺。最后，由于FTIR在高浓度（因此大多处于饱和状态）下工作，它能清楚地将对亲和力的影响与纯粹的催化修饰区分开来，表明在RapGAP之间保守且在同源的RheBGAP结节蛋白中发生突变的精氨酸388会严重影响结合亲和力，但对裂解反应本身没有影响。
