SUBSTRATE AND PRODUCT STRUCTURAL REQUIREMENTS FOR BINDING OF NUCLEOTIDES TO H-RAS P21 - THE MECHANISM OF DISCRIMINATION BETWEEN GUANOSINE AND ADENOSINE NUCLEOTIDES
SUBSTRATE AND PRODUCT STRUCTURAL REQUIREMENTS FOR BINDING OF NUCLEOTIDES TO H-RAS P21 - THE MECHANISM OF DISCRIMINATION BETWEEN GUANOSINE AND ADENOSINE NUCLEOTIDES
复制标题
DOI：
10.1021/bi00002a026
复制
发表时间：
1995-01-17
期刊：
影响因子：
2.9
通讯作者：
GOODY, RS
中科院分区：
文献类型：
--
作者：
RENSLAND, H;JOHN, J;GOODY, RS
H-ras癌基因的蛋白产物与一系列核苷二磷酸和三磷酸的相互作用已被检查，以研究活性位点对GTP或GDP结构偏离的耐受性。结合力较强的核苷酸可在简单的滤膜结合试验中用作GDP的竞争者，以半定量地估计它们的亲和力。对于更弱结合的核苷酸或获得定量数据，使用基于测定缔合和解离速率常数的瞬时动力学方法。所获得的结果表明，糖或磷酸盐结构的实质性修饰是耐受的，具有很少或中等的亲和力损失，但是在碱基结构的修饰上发生亲和力的大损失。特别地，用腺嘌呤残基替换鸟嘌呤导致亲和力的显著损失。因此，对ATP和ADP的区分度非常高（ATP和GTP的相对亲和力为1：10（7））。这不仅是因为失去了积极的（稳定的）相互作用，而且特别是因为引入了消极的相互作用。
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.