Allosteric regulation of the class III anaerobic ribonucleotide reductase from bacteriophage T4
Allosteric regulation of the class III anaerobic ribonucleotide reductase from bacteriophage T4
复制标题
DOI：
10.1074/jbc.m001490200
复制
发表时间：
2000-06-30
影响因子：
4.8
通讯作者：
Sjöberg, BM
中科院分区：
文献类型：
--
作者：
Andersson, J;Westman, M;Sjöberg, BM
核糖核苷酸还原酶（Ribonucleotide reductase，RNR）是所有生物体必需的酶，它通过将四种核糖核苷酸还原为脱氧核糖核苷酸来提供DNA合成的前体。RNR的总活性和底物特异性受三磷酸脱氧核糖核苷和ATP的别构调节，从而提供平衡的dNTP库。我们的研究结果表明，T4酶有一个单一类型的变构位点，dGTP，dTTP，dATP和ATP竞争性结合。解离常数在微摩尔范围内，除了ATP，其解离常数在毫摩尔范围内。ATP和dATP是CTP减少的正效应物，dGTP是ATP减少的正效应物，dTTP是GTP减少的正效应物。dATP不是一般的负变构效应物，这些效应类似于Ib类和II类RNR的变构调节，以及噬菌体T4的Ia类RNR，但不同于来自宿主细菌大肠杆菌和乳酸乳球菌的III类RNR。其模拟了生理情况并说明了体内不同效应物之间的相互作用。令人惊讶的是，我们在所用条件下没有观察到任何显著的UTP减少。嘧啶脱氧核糖核苷酸库的平衡可以通过dCMP脱氨酶和dCMP羟甲基化酶途径实现。
Ribonucleotide reductase (RNR) is an essential enzyme in all organisms, It provides precursors for DNA synthesis by reducing all four ribonucleotides to deoxyribonucleotides, The overall activity and the substrate specificity of RNR are allosterically regulated by deoxyribonucleoside triphosphates and ATP, thereby providing balanced dNTP pools, We have characterized the allosteric regulation of the class III RNR from bacteriophage T4. Our results show that the T4 enzyme has a single type of allosteric site to which dGTP, dTTP, dATP, and ATP bind competitively. The dissociation constants are in the micromolar range, except for ATP, which has a dissociation constant in the millimolar range. ATP and dATP are positive effecters for CTP reduction, dGTP is a positive effector for ATP reduction, and dTTP is a positive effector for GTP reduction. dATP is not a general negative allosteric effector, These effects are similar to the allosteric regulation of class Ib and class II RNRs, and to the class Ia RNR of bacteriophage T4, but differ from that of the class III RNRs from the host bacterium Escherichia coli and from Lactococcus lactis.The relative rate of reduction of the four substrates was measured simultaneously in a mixed-substrate assay, which mimics the physiological situation and illustrates the interplay between the different effecters in vivo Surprisingly, we did not observe any significant UTP reduction under the conditions used, Balancing of the pyrimidine deoxyribonucleotide pools may be achieved via the dCMP deaminase and dCMP hydroxymethylase pathways.