The mechanism of adenosylmethionine-dependent activation of methionine synthase: A rapid kinetic analysis of intermediates in reductive methylation of cob(II)alamin enzyme
The mechanism of adenosylmethionine-dependent activation of methionine synthase: A rapid kinetic analysis of intermediates in reductive methylation of cob(II)alamin enzyme
复制标题
DOI：
10.1021/bi9808565
复制
发表时间：
1998-09-08
期刊：
影响因子：
2.9
通讯作者：
Matthews, RG
中科院分区：
文献类型：
--
作者：
Jarrett, JT;Hoover, DM;Matthews, RG
依赖钴胺的蛋氨酸合成酶催化甲基四氢叶酸向同型半胱氨酸转移，生成四氢叶酸和蛋氨酸。在这个主要的周转周期中，该酶在活性的甲钴胺和CoB(I)丙氨酸酶之间交替。由于CoB(I)丙胺的氧化或甲钴胺的光解而形成CoB(II)丙氨酸基，使该酶失活。来自大肠杆菌的蛋氨酸合成酶通过还原甲基化来催化自身的重新激活，该甲基化涉及到来自还原黄毒素的电子转移和来自ADOMet的甲基转移。这一过程被认为涉及形成一种瞬时的CoB(I)丙胺中间体，然后被来自Adobe Met的甲基转移捕获。在大肠杆菌的有氧生长过程中，这个过程的电子最终来自NADPH，并且由于NADPH/NADP(+)对和CoB(I)Alamin/Cob(II)Alamin对之间的巨大电势差，电子转移不会产生可检测到的Cob(I)Alamin水平。在这篇文章中，我们证明了即使在强还原剂黄曲霉毒素对苯二酚的存在下，CoB(I)丙胺也没有被观察到作为一个重要的中间体。然而，我们证明这是由于钴配体环境从五配位到四配位的限速重组所致。天冬氨酸757突变为谷氨酸导致CoB(II)丙氨酸酶类似于70%的四配位，该酶的还原甲基化通过具有动力学活性的CoB(I)丙氨酸中间体进行。此外，化学还原产生的野生型CoB(I)丙氨酸酶与ADOMet发生动力学反应。我们提供的证据表明，甲基化从ADOMet转移到CoB(I)丙氨酸酶最初会导致五配位甲钴胺酶的形成，该酶会慢慢降解为活性的六配位甲钴胺酶。我们提出了腺苷蛋氨酸和黄曲霉毒素对苯二酚还原甲基化野生型CoB(II)丙氨酸酶的动力学方案，其中缓慢的构象变化掩盖了相对较快的电子和甲基转移步骤。
Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl g-roup from methyltetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. During this primary turnover cycle, the enzyme alternates between the active methylcobalamin and cob(I)alamin forms of the enzyme. Formation of the cob(II)alamin prosthetic group by oxidation of cob(I)alamin or photolysis of methylcobalamin renders the enzyme inactive. Methionine synthase from E. coli catalyzes its own reactivation by a reductive methylation that involves electron transfer from reduced flavodoxin and methyl transfer from AdoMet. This process has been proposed to involve formation of a transient cob(I)alamin intermediate that is then trapped by methyl transfer from AdoMet. During aerobic growth of E. coli, electrons for this process are ultimately derived from NADPH, and electron transfer does not generate a detectable level of cob(I)alamin due to the large potential difference between the NADPH/NADP(+) couple and the cob(I)alamin/cob(II)alamin couple. In this paper, we show that even in the presence of the strong reductant flavodoxin hydroquinone, cob(I)alamin is not observed as a significant intermediate. We demonstrate, however, that this is due to a rate-limiting reorganization of the cobalt ligand environment from five-coordinate to four-coordinate cob(II)alamin. Mutation of aspartate 757 to glutamate results in a cob(II)alamin enzyme that is similar to 70% four-coordinate, and reductive methylation of this enzyme using flavodoxin hydroquinone as the electron donor proceeds through a kinetically competent cob(I)alamin intermediate. Furthermore, wild-type cob(I)alamin enzyme produced by chemical reduction reacts with AdoMet in a kinetically competent reaction. We provide evidence that methyl transfer from AdoMet to cob(I)alamin enzyme results initially in formation of a five-coordinate methylcobalamin enzyme that slowly decays to the active six-coordinate methylcobalamin enzyme. We propose a kinetic scheme for reductive methylation of wild-type cob(II)alamin enzyme by adenosylmethionine and flavodoxin hydroquinone in which slow conformational changes mask the relatively fast electron and methyl transfer steps.