Yamanishi M, Yamada S, Muguruma H, Murakami Y, Tobimatsu T, Ishida A, Yamauchi J, Toraya T
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700, Japan.
Biochemistry. 1998 Apr 7;37(14):4799-803. doi: 10.1021/bi972572a.
It was demonstrated by electron paramagnetic resonance (EPR) spectroscopy that organic radical intermediates disappeared and cob(II)alamin accumulated upon suicide inactivation of diol dehydratase by 2-methyl-1,2-propanediol. The resulting EPR spectra showed that the eight hyperfine lines due to the divalent cobalt atom of cob(II)alamin further split into triplets by the superhyperfine coupling to the 14N nucleus. Essentially the same superhyperfine splitting of the octet into triplets was observed with [14N]- and [15N]apoenzyme. When the adenosyl form of [14N2]- and [15N2]imidazolyl analogues of the coenzyme [Toraya, T., and Ishida, A. (1991) J. Biol. Chem. 266, 5430-5437] was used with unlabeled apoenzyme, the octet showed superhyperfine splitting into triplets and doublets, respectively. Therefore, it was concluded that cobalamin is bound to this enzyme with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. This conclusion is consistent with the fact that the consensus sequence forming part of a cobalamin-binding motif, conserved in methionine synthase and some of the other cobalamin enzymes, was not found in the deduced amino acid sequences of the subunits of diol dehydratase. Adenosylcobinamide methyl phosphate, a coenzyme analogue lacking the nucleotide moiety, underwent cleavage of the cobalt-carbon bond upon binding to the enzyme in the presence of substrate, forming a cob(II)inamide derivative without nitrogenous base coordination, as judged by EPR and optical spectroscopy. Therefore, this analogue may be a useful probe for determining whether the replacement of the 5, 6-dimethylbenzimidazole ligand by a histidine residue takes place upon binding of cobalamin to proteins.
电子顺磁共振（EPR）光谱表明，2-甲基-1,2-丙二醇使二醇脱水酶自杀失活后，有机自由基中间体消失，而钴胺素（II）积累。所得的EPR光谱显示，钴胺素（II）中二价钴原子产生的八条超精细线通过与14N核的超超精细偶合进一步分裂为三重态。在[14N]-和[15N]脱辅基酶中观察到了八重态基本相同的分裂为三重态的超超精细分裂。当辅酶的[14N2]-和[15N2]咪唑基类似物的腺苷形式[Toraya, T., and Ishida, A. (1991) J. Biol. Chem. 266, 5430-5437]与未标记的脱辅基酶一起使用时，八重态分别显示出分裂为三重态和二重态的超超精细分裂。因此，可以得出结论，钴胺素与该酶结合时，5,6-二甲基苯并咪唑与钴原子配位。这一结论与以下事实一致：在甲硫氨酸合酶和其他一些钴胺素酶中保守的构成钴胺素结合基序一部分的共有序列，在二醇脱水酶亚基的推导氨基酸序列中未发现。缺乏核苷酸部分的辅酶类似物腺苷钴胺素甲基磷酸，在底物存在下与酶结合时发生钴-碳键的断裂，通过EPR和光谱学判断，形成了没有含氮碱基配位的钴胺素（II）酰胺衍生物。因此，这种类似物可能是一种有用的探针，用于确定钴胺素与蛋白质结合时，5,6-二甲基苯并咪唑配体是否被组氨酸残基取代。
