Epimerization at carbon-5′ of (5′R)-[5′,2H]adenosylcobalamin by ribonucleoside triphosphate reductase:: Cysteine 408-independent cleavage of the Co-C5′ bond
Epimerization at carbon-5′ of (5′R)-[5′,2H]adenosylcobalamin by ribonucleoside triphosphate reductase:: Cysteine 408-independent cleavage of the Co-C5′ bond
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DOI：
10.1021/bi030018x
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发表时间：
2003-04-22
期刊：
影响因子：
2.9
通讯作者：
Frey, PA
中科院分区：
文献类型：
--
作者：
Chen, DW;Abend, A;Frey, PA
来自莱氏乳杆菌的腺苷钴胺素依赖性核糖核苷三磷酸还原酶（RTPR）催化核糖核苷三磷酸还原为脱氧核糖核苷三磷酸。RTPR还催化腺苷钴胺素的C5 '-氢与溶剂氢的交换。位于Cys 408上的硫基自由基是通过腺苷钴胺素在活性位点的反应产生的，并且被认为是核苷酸还原和5 '-氢交换反应的中间体。本文采用立体化学方法研究了腺苷钴胺素在RTPR反应中Co-C5'键断裂的机理。当立体选择性氘代辅酶（5 ′ R）-[5 ′-H-2（1）]腺苷钴胺素（5 ′ R/S = 3：1）与RTPR或Cys 408变体C408 A-RTPR和C408 S-RTPR在dGTP存在下孵育时，5 ′-碳上的氘在立体化学上被扰乱，导致（5 ′ S）-[5 ′-H-2（1）]-和（5 ′ R）-[5 ′-H-2（1）]-同位素异构体的差向异构化。用突变的RTPR观察差向异构化证明Co-C5'键的瞬时裂解在Cys 408上不存在巯基的情况下发生。在dGTP存在下，RTPR、C408 A-RTPR和C408 S-RTPR的差向异构化速率常数分别为5.1、0.28和0.42 s（-1）。只有野生型RTPR催化5 '-氢交换反应。差向异构化和5 '-氢交换反应都受到变构效应物dGTP的刺激，并且在不存在效应物的情况下未检测到差向异构化。机制方面的影响wt-RTPR介导的碳钴键均裂和中间的5 '-脱氧腺苷自由基。
The adenosylcobalamin-dependent ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of ribonucleoside triphosphates to deoxyribonucleoside triphosphates. RTPR also catalyzes the exchange of the C5'-hydrogens of adenosylcobalalamin with solvent hydrogen. A thiyl radical located on Cys 408 is generated by reaction of adenosylcobalamin at the active site and is proposed to be the intermediate for both the nucleotide reduction and the 5'-hydrogen exchange reactions. In the present research, a stereochemical approach is used to study the mechanism of the Co-C5' bond cleavage of adenosylcobalamin in the reaction of RTPR. When stereoselectively deuterated coenzyme, (5'R)-[5'-H-2(1)] adenosylcobalamin (5'R/S = 3:1), was incubated with RTPR or the Cys 408 viariants, C408A-RTPR and C408S-RTPR in the presence of dGTP, the deuterium at the 5'-carbon was stereochemically scrambled, leading to epimerization of the (5'S)-[5'-H-2(1)]- and (5R)-[5'-H-2(1)]-isotopomers. Observation of epimerization with mutated RTPR proves that transient cleavage of the Co-C5' bond occurs in the absence of the thiol group on Cys 408. The rate constants for epimerization by RTPR, C408A-RTPR, and C408S-RTPRs in the presence of dGTP are 5.1, 0.28, and 0.42 s(-1), respectively. Only the wild-type RTPR catalyzes the 5'-hydrogen exchange reaction. Both epimerization and 5'-hydrogen exchange reactions are stimulated by the allosteric effector dGTP, and epimerization is not detected in the absence of the effector. Mechanistic implications with respect to wt-RTPR-mediated carbon cobalt bond homolysis and the intermediacy of the 5'-deoxyadenosyl radical will be presented.