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首页> 外文期刊>Journal of Molecular Biology >Role of arginine 293 and glutamine 288 in communication between catalytic and allosteric sites in yeast ribonucleotide reductase
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Role of arginine 293 and glutamine 288 in communication between catalytic and allosteric sites in yeast ribonucleotide reductase

机译：精氨酸293和谷氨酰胺288在酵母核糖核苷酸还原酶催化位点和变构位点之间的通讯中的作用

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Ribonucleotide reductases (RRs) catalyze the rate-limiting step of de novo deoxynucleotide (dNTP) synthesis. Eukaryotic RRs consist of two proteins, RR1 (α) that contains the catalytic site and RR2 (β) that houses a diferric-tyrosyl radical essential for ribonucleoside diphosphate reduction. Biochemical analysis has been combined with isothermal titration calorimetry (ITC), X-ray crystallography and yeast genetics to elucidate the roles of two loop 2 mutations R293A and Q288A in Saccharomyces cerevisiae RR1 (ScRR1). These mutations, R293A and Q288A, cause lethality and severe S phase defects, respectively, in cells that use ScRR1 as the sole source of RR1 activity. Compared to the wild-type enzyme activity, R293A and Q288A mutants show 4% and 15%, respectively, for ADP reduction, whereas they are 20% and 23%, respectively, for CDP reduction. ITC data showed that R293A ScRR1 is unable to bind ADP and binds CDP with 2-fold lower affinity compared to wild-type ScRR1. With the Q288A ScRR1 mutant, there is a 6-fold loss of affinity for ADP binding and a 2-fold loss of affinity for CDP compared to the wild type. X-ray structures of R293A ScRR1 complexed with dGTP and AMPPNP-CDP [AMPPNP, adenosine 5-(β,γ-imido)triphosphate tetralithium salt] reveal that ADP is not bound at the catalytic site, and CDP binds farther from the catalytic site compared to wild type. Our in vivo functional analyses demonstrated that R293A cannot support mitotic growth, whereas Q288A can, albeit with a severe S phase defect. Taken together, our structure, activity, ITC and in vivo data reveal that the arginine 293 and glutamine 288 residues of ScRR1 are crucial in facilitating ADP and CDP substrate selection.
机译：核糖核苷酸还原酶（RRs）催化从头进行脱氧核苷酸（dNTP）合成的限速步骤。真核生物的RRs由两种蛋白质组成，RR1（α）含有催化位点，RR2（β）含有对二磷酸核糖核苷还原至关重要的二铁-酪氨酰基。生化分析已与等温滴定热法（ITC），X射线晶体学和酵母遗传学相结合，阐明了啤酒酵母RR1（ScRR1）中两个loop 2突变R293A和Q288A的作用。在使用ScRR1作为RR1活性唯一来源的细胞中，这些突变R293A和Q288A分别导致致死性和严重的S期缺陷。与野生型酶活性相比，R293A和Q288A突变体的ADP降低分别为4％和15％，而CDP降低则分别为20％和23％。 ITC数据显示，与野生型ScRR1相比，R293A ScRR1无法结合ADP并以低2倍的亲和力结合CDP。与野生型相比，Q288A ScRR1突变体对ADP结合的亲和力损失了6倍，对CDP的亲和力损失了2倍。 R293A ScRR1与dGTP和AMPPNP-CDP复合的X射线结构[AMPPNP，腺苷5-（β，γ-亚氨基）三磷酸四锂盐]显示ADP在催化位点未结合，而CDP结合更远离催化位点与野生型相比。我们的体内功能分析表明，R293A不能支持有丝分裂的生长，而Q288A却可以支持严重的S期缺损。综上所述，我们的结构，活性，ITC和体内数据表明，ScRR1的精氨酸293和谷氨酰胺288残基对于促进ADP和CDP底物的选择至关重要。

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