Enzymatic activity of coenzyme B(12) derivatives with altered axial nucleotides: probing the mechanochemical triggering hypothesis in ribonucleotide reductase.
Enzymatic activity of coenzyme B(12) derivatives with altered axial nucleotides: probing the mechanochemical triggering hypothesis in ribonucleotide reductase.
复制标题
轴向核苷酸改变的辅酶 B(12) 衍生物的酶活性：探讨核糖核苷酸还原酶的机械化学触发假说。
DOI：
10.1021/ic010796i
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发表时间：
2001
影响因子：
4.6
通讯作者：
Chen,G
中科院分区：
文献类型：
--
作者：
Brown,KL;Zou,X;Li,J;Chen,G
Theoretical studies (J. Inorg. Biochem.2001,83, 121) of the involvement of the bulky 5,6-dimethylbenzimidazole (Dmbz) ligand of coenzyme B12(5‘-deoxyadenosylcobalamin, AdoCbl) in the mechanism of activation of the carbon−cobalt bond of the coenzyme for homolytic cleavage by AdoCbl-dependent enzymes (the “mechanochemical triggering” mechanisms) have shown that a purely steric, ground-state mechanism can supply only a few kilocalories per mole (of the observed 13−16 kcal mol-1) of activation, but that an electronic mechanism, operating to stabilize the transition state, can explain all of the observed catalytic effect. To address these mechanisms experimentally, analogues of AdoCbl in which the Dmbz ligand is replaced by benzimidazole (Ado(Bzim)Cbl) or by imidazole (Ado(Im)Cbl) have been prepared and characterized. Both of these analogues support turnover in the AdoCbl-dependent ribonucleoside triphosphate reductase (RTPR) fromLactobacillus leichmanniiat 100% of the activity of AdoCbl itself, but the Ado(Im)Cbl analogue has a significantly higherKm. 5‘-Deoxyadenosylcobinamide, the analogue in which the axial nucleotide has been chemically removed, in contrast, is inactive in the spectrophotometric assay, which indicates that it has at most 1% of the activity of AdoCbl. Stopped-flow spectrophotometric measurements of the formation of cob(II)alamin at the enzyme active site show that RTPR binds Ado(Bzim)Cbl slightly more weakly than it does AdoCbl, but binds Ado(Im)Cbl 8-fold more weakly. While the equilibrium constant for cob(II)alamin formation is nearly the same for Ado(Bzim)Cbl and AdoCbl, it is 5-fold smaller for Ado(Im)Cbl. Finally, the forward rate constant for enzyme-induced Co−C bond homolysis was about the same for Ado(Bzim)Cbl and for AdoCbl but was 17-fold smaller for Ado(Im)Cbl. These results are consistent with a small contribution from ground-state mechanochemical triggering, but they do not in themselves rule out transition-state mechanical triggering.
