Toraya T, Mori K
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700-8530, Japan.
J Biol Chem. 1999 Feb 5;274(6):3372-7. doi: 10.1074/jbc.274.6.3372.
Adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicide inactivation by glycerol, a physiological substrate. The coenzyme is modified through irreversible cleavage of its cobalt-carbon bond, resulting in inactivation of the enzyme by tight binding of the modified coenzyme to the active site. Recombinant DdrA and DdrB proteins of K. oxytoca were co-purified to homogeneity from cell-free extracts of Escherichia coli overexpressing the ddrAB genes. They existed as a tight complex, i.e. a putative reactivating factor, with an apparent molecular weight of 150,000. The factor consists of equimolar amounts of the two subunits with Mr of 64,000 (A) and 14,000 (B), encoded by the ddrA and ddrB genes, respectively. Therefore, its subunit structure is most likely A2B2. The factor not only reactivated glycerol-inactivated and O2-inactivated holoenzymes but also activated enzyme-cyanocobalamin complex in the presence of free adenosylcobalamin, ATP, and Mg2+. The reactivating factor mediated ATP-dependent exchange of the enzyme-bound cyanocobalamin for free 5-adeninylpentylcobalamin in the presence of ATP and Mg2+, but the reverse was not the case. Thus, it can be concluded that the inactivated holoenzyme becomes reactivated by exchange of the enzyme-bound, adenine-lacking cobalamins for free adenosylcobalamin, an adenine-containing cobalamin.
产酸克雷伯菌的腺苷钴胺素依赖性二醇脱水酶会被其生理底物甘油进行自杀性失活。辅酶通过其钴-碳键的不可逆断裂而被修饰，导致修饰后的辅酶紧密结合到活性位点，从而使酶失活。产酸克雷伯菌的重组DdrA和DdrB蛋白从过量表达ddrAB基因的大肠杆菌无细胞提取物中共同纯化至同质。它们以紧密复合物的形式存在，即一种假定的再激活因子，表观分子量为150,000。该因子由等摩尔量的两个亚基组成，Mr分别为64,000（A）和14,000（B），分别由ddrA和ddrB基因编码。因此，其亚基结构最可能是A2B2。该因子不仅能使甘油失活和氧气失活的全酶重新激活，还能在游离腺苷钴胺素、ATP和Mg2+存在的情况下激活酶-氰钴胺复合物。在ATP和Mg2+存在的情况下，再激活因子介导酶结合的氰钴胺与游离的5-腺苷基戊基钴胺素进行ATP依赖性交换，但反之则不然。因此，可以得出结论，失活的全酶通过将酶结合的、缺乏腺嘌呤的钴胺素与游离的腺苷钴胺素（一种含腺嘌呤的钴胺素）进行交换而重新激活。
