核糖核苷三磷酸还原酶催化碳-钴键均裂的研究：协同碳-钴键均裂和硫自由基形成的证据。

Studies on the catalysis of carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase: evidence for concerted carbon-cobalt bond homolysis and thiyl radical formation.

作者信息

Licht S S, Booker S, Stubbe J

机构信息

Departments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

出版信息

Biochemistry. 1999 Jan 26;38(4):1221-33. doi: 10.1021/bi981885i.

DOI:10.1021/bi981885i

PMID:9930982

Abstract

Ribonucleotide reductases (RNRs) catalyze the rate-determining step in DNA biosynthesis: conversion of nucleotides to deoxynucleotides. The RNR from Lactobacillus leichmannii utilizes adenosylcobalamin (AdoCbl) as a cofactor and, in addition to nucleotide reduction, catalyzes the exchange of tritium from [5'-3H]-AdoCbl with solvent. Examination of this exchange reaction offers a unique opportunity to investigate the early stages in the nucleotide reduction process [Licht S. S., Gerfen, G. J., and Stubbe, J. (1996) Science 271, 477-481]. The kinetics of and requirements for this exchange reaction have been examined in detail. The turnover number for 3H washout is 0.3 s-1, and it requires an allosteric effector dGTP (Km = 17 +/- 3 microM), AdoCbl (Km = 60 +/- 9 microM) and no external reductant. The effects of active-site mutants of RTPR (C119S, C419S, C731S, C736S, and C408S) on the rate of the exchange reaction have been determined, and only C408 is essential for this process. The exchange reaction has previously been monitored by stopped-flow UV-vis spectroscopy, and cob(II)alamin was shown to be formed with a rate constant of 40 s-1 [Tamao, Y., and Blakley, R. L. (1973) Biochemistry 12, 24-34]. This rate constant has now been measured in D2O, with [5'-2H2]-AdoCbl in H2O, and with [5'-2H2]-AdoCbl in D2O. A comparison of these results with those for AdoCbl in H2O revealed kH/kD of 1.6, 1.7, and 2.7, respectively. The absolute amounts of cob(II)alamin generated with [5'-2H2]-AdoCbl in D2O in comparison with AdoCbl in H2O reveal twice as much cob(II)alamin in the former case. Similar transient kinetic studies with C408S RTPR reveal no cob(II)alamin formation. These experiments allow proposal of a minimal mechanism for this exchange reaction in which RNR catalyzes homolysis of the carbon-cobalt bond in a concerted fashion, to generate a thiyl radical on C408, cob(II)alamin, and 5'-deoxyadenosine.

摘要

核糖核苷酸还原酶（RNRs）催化DNA生物合成中的限速步骤：核苷酸转化为脱氧核苷酸。来自莱氏乳杆菌的RNR利用腺苷钴胺素（AdoCbl）作为辅因子，除了核苷酸还原外，还催化[5'-3H]-AdoCbl中的氚与溶剂的交换。对这种交换反应的研究为探究核苷酸还原过程的早期阶段提供了独特的机会[利希特·S.S.、格芬·G.J.和斯塔布·J.（1996年）《科学》271卷，477 - 481页]。已经详细研究了这种交换反应的动力学和条件。3H洗脱的周转数为0.3 s-1，它需要变构效应物dGTP（Km = 17 ± 3 microM）、AdoCbl（Km = 60 ± 9 microM）且不需要外部还原剂。已经确定了RTPR活性位点突变体（C119S、C419S、C731S、C736S和C408S）对交换反应速率的影响，只有C408对该过程至关重要。以前通过停流紫外可见光谱监测交换反应，结果表明生成钴胺素（II）的速率常数为40 s-1[玉尾洋、布莱克利·R.L.（1973年）《生物化学》12卷，24 - 34页]。现在已经在D2O中、在H2O中用[5'-2H2]-AdoCbl以及在D2O中用[5'-2H2]-AdoCbl测量了这个速率常数。将这些结果与在H2O中AdoCbl的结果进行比较，分别得到kH/kD为1.6、1.7和2.7。与在H2O中的AdoCbl相比，在D2O中用[5'-2H2]-AdoCbl生成的钴胺素（II）的绝对量表明，在前一种情况下生成的钴胺素（II）是后者的两倍。用C408S RTPR进行的类似瞬态动力学研究表明没有生成钴胺素（II）。这些实验使得能够提出这种交换反应的最小机制，即RNR以协同方式催化碳 - 钴键的均裂，在C408上生成硫自由基、钴胺素（II）和5'-脱氧腺苷。