Importance of F1-ATPase residue α-Arg-376 for catalytic transition state stabilization
Importance of F1-ATPase residue α-Arg-376 for catalytic transition state stabilization
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DOI：
10.1021/bi9917683
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发表时间：
1999-11-23
期刊：
影响因子：
2.9
通讯作者：
Senior, AE
中科院分区：
文献类型：
--
作者：
Nadanaciva, S;Weber, J;Senior, AE
研究了必需残基α-Arg-376在F-1-ATPase催化部位的功能作用。构建了突变体αR376C、αR376Q和αR376K，并与突变βY331W结合，研究了催化位点的核苷酸结合参数，并通过测量镁ADP与氟铝酸盐的结合来评估催化过渡态的形成。每一个突变都会对ATP的合成和降解造成很大的损害。尽管在已发表的X射线结构中，α-Arg-376与核苷二磷酸和三磷酸结合很近，但突变对镁ADP或镁ATP结合亲和力几乎没有影响，特别是在亲和力最高的催化位点1。Cys和Gin突变体都取消了过渡态的形成，表明α-Arg-376通常参与了这一催化步骤。提出并讨论了F-1-ATPase催化过渡态结构模型。Lys突变体虽然严重受损，但支持过渡态的形成，这表明α-Arg-376胍基还存在一个重要的作用，可能是在稳态催化所需的α/β构象信号传递中。α-Arg-376和GAP/G蛋白“精氨酸指”残基之间的相似之处是显而易见的。
The functional role of essential residue alpha-Arg-376 in the catalytic site of F-1-ATPase was studied. The mutants alpha R376C, alpha R376Q, and alpha R376K were constructed, and combined with the mutation beta Y331W, to investigate catalytic site nucleotide-binding parameters, and to assess catalytic transition state formation by measurement of MgADP-fluoroaluminate binding. Each mutation caused large impairment of ATP synthesis and hydrolysis. Despite the apparent proximity of alpha-Arg-376 to bound nucleoside di- and triphosphate in published X-ray structures, the mutations had little effect on MgADP or MgATP binding affinities, particularly at the highest affinity catalytic site, site 1. Both Cys and Gin mutants abolished transition state formation, demonstrating that alpha-Arg-376 is normally involved at this step of catalysis. A model of the F-1-ATPase catalytic transition state structure is presented and discussed. The Lys mutant, although severely impaired, supported transition state formation, suggesting that an additional essential role for the alpha-Arg-376 guanidinium group exists, likely in alpha/beta conformational signal transmission required for steady-state catalysis. Parallels between alpha-Arg-376 and GAP/G-protein "arginine finger" residues are evident.