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首页> 外文期刊>Biochemistry >Substrate and product structural requirements for binding of nucleotides to H-ras p21: the mechanism of discrimination between guanosine and adenosine nucleotides.
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Substrate and product structural requirements for binding of nucleotides to H-ras p21: the mechanism of discrimination between guanosine and adenosine nucleotides.

机译:核苷酸与H-ras p21结合的底物和产品结构要求:鸟苷和腺苷核苷酸之间区别的机理。

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摘要

The interaction of the protein product of the H-ras oncogene with a series of nucleoside di- and triphosphates has been examined to investigate the tolerance of the active site to departures from the GTP or GDP structures. Nucleotides which bind relatively strongly could be used as competitors of GDP in a simple filter binding assay to give semiquantitave estimates of their affinities. For more weakly binding nucleotides or to obtain quantitative data, a transient kinetic method was used which was based on determination of the association and dissociation rate constants. The results obtained indicate that substantial modification of the sugar or phosphate structure is tolerated with little or moderate loss of affinity, but that large losses in affinity occur on modification of the base structure. In particular, replacing the guanine by an adenine residue leads to a dramatic loss of affinity. Thus, discrimination against ATP and ADP is very high (relative affinities of ATP and GTP 1:10(7)). This is due not only to loss of positive (stabilizing) interactions, but especially to the introduction of negative ones.
机译:已经检查了H-ras癌基因的蛋白质产物与一系列核苷二磷酸和三磷酸的相互作用,以研究活性位点对偏离GTP或GDP结构的耐受性。在一个简单的过滤器结合试验中,结合力相对较强的核苷酸可以用作GDP的竞争者,从而得出其亲和力的半定量估计值。为了更弱地结合核苷酸或获得定量数据,使用了基于确定缔合和解离速率常数的瞬态动力学方法。所获得的结果表明,糖或磷酸酯结构的实质性修饰可以忍受很少或中等的亲和力损失,但是在基础结构的修饰上亲和力的损失却很大。特别地,用腺嘌呤残基代替鸟嘌呤导致亲和力的显着丧失。因此,对ATP和ADP的辨别率很高(ATP和GTP的相对亲和力为1:10(7))。这不仅是由于失去了积极的(稳定的)相互作用,而且还特别是由于引入了消极的相互作用。

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