化学
核苷酸
核苷酸还原酶
反应速率常数
辅因子
立体化学
蛋白质亚单位
离解(化学)
核苷酸
动力学
生物物理学
生物化学
酶
有机化学
生物
基因
物理
量子力学
作者
Kanchana R. Ravichandran,Lisa Olshansky,Daniel G. Nocera,Jo Anne Stubbe
出处
期刊:Biochemistry
[American Chemical Society]
日期:2020-03-18
卷期号:59 (14): 1442-1453
被引量:10
标识
DOI:10.1021/acs.biochem.0c00001
摘要
Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDP) to deoxynucleotides (dNDP), in part, by controlling the ratios and quantities of dNTPs available for DNA replication and repair. The active form of Escherichia coli class Ia RNR is an asymmetric α2β2 complex in which α2 contains the active site and β2 contains the stable diferric-tyrosyl radical cofactor responsible for initiating the reduction chemistry. Each dNDP is accompanied by disulfide bond formation. We now report that, under in vitro conditions, β2 can initiate turnover in α2 catalytically under both “one” turnover (no external reductant, though producing two dCDPs) and multiple turnover (with an external reductant) assay conditions. In the absence of reductant, rapid chemical quench analysis of a reaction of α2, substrate, and effector with variable amounts of β2 (1-, 10-, and 100-fold less than α2) yields 3 dCDP/α2 at all ratios of α2:β2 with a rate constant of 8–9 s–1, associated with a rate-limiting conformational change. Stopped-flow fluorescence spectroscopy with a fluorophore-labeled β reveals that the rate constants for subunit association (163 ± 7 μM–1 s–1) and dissociation (75 ± 10 s–1) are fast relative to turnover, consistent with catalytic β2. When assaying in the presence of an external reducing system, the turnover number is dictated by the ratio of α2:β2, their concentrations, and the concentration and nature of the reducing system; the rate-limiting step can change from the conformational gating to a step or steps involving disulfide rereduction, dissociation of the inhibited α4β4 state, or both. The issues encountered with E. coli RNR are likely of importance in all class I RNRs and are central to understanding the development of screening assays for inhibitors of these enzymes.
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