Kreft J U, Schink B
Fakultät für Biologie, Universität Konstanz, Germany.
Eur J Biochem. 1994 Dec 15;226(3):945-51. doi: 10.1111/j.1432-1033.1994.00945.x.
The previously studied complete methyl transfer sequence of tetrahydrofolate-dependent O-demethylation catalyzed by Holophaga foetida strain TMBS4 extracts was separated into two steps using cobalamins as non-physiological substrates: electrochemically produced cob(I) alamin served as methyl acceptor for phenyl methyl ether demethylation, yielding methylcob(III)alamin (reaction I), and methylcob(III)alamin served as donor for tetrahydrofolate methylation, yielding 5-methyl tetrahydrofolate (reaction II). Both reactions were measured with a new and direct photometric assay of cob(I)alamin methylation (or the reverse reaction) at 540 nm, the isobestic wavelength of the cob(II)alamin/cob(I)alamin redox couple (delta epsilon 540 = 4.40 nM-1.cm-1. The rates of reactions I and II were proportional to protein concentration, unlike the complete reaction sequence. Small components of cell extract did not affect activity of reactions I and II. Isovanillate demethylation by extracts of synringate-grown cells (reaction I) required reductive activation by cob(I)alamin and was inhibited and inactivated by cob(II)alamin, indicating that the reaction mechanism was a nucleophilic attack of an enzyme-bound corrinoid in the reduced Co(I) state on the methyl carbon of the ether, rather than a radical attack. Only phenyl methyl ethers were demethylated; demethylation rates were enhanced by ortho-hydroxyl or para-carboxyl groups, but reduced by additional meta substituents. The rate of isovanillate demethylation was 81 nmol.min-1.(mg protein)-1 [0.76 mM cob(I)alamin] and apparent kinetic constants for cob(I)alamin were: Km = 1.2 mM, Vmax = 220 nmol min-1.(mg protein)-1, and Vmax/Km = 180 nmol.min-1.(mg protein) 1.mM-1 3,5-Dihydroxyanisole demethylation by extracts of 3,5-dihydroxyanisole-grown cells (also reaction I) was much slower. Reaction II did not require activation; specific activity and the specificity constant for methylcob(III)alamin were much lower.
先前对恶臭食菌菌株TMBS4提取物催化的依赖四氢叶酸的O-去甲基化完整甲基转移序列进行的研究，使用钴胺素作为非生理性底物将其分为两个步骤：电化学产生的钴胺素（I）作为苯甲醚去甲基化的甲基受体，生成甲基钴胺素（III）（反应I），甲基钴胺素（III）作为四氢叶酸甲基化的供体，生成5-甲基四氢叶酸（反应II）。这两个反应均通过一种新的直接光度法测定钴胺素（I）甲基化（或逆反应），在540nm处进行测量，540nm是钴胺素（II）/钴胺素（I）氧化还原对的等吸收波长（Δε540 = 4.40nM-1·cm-1）。与完整反应序列不同，反应I和II的速率与蛋白质浓度成正比。细胞提取物中的小成分不影响反应I和II的活性。丁香酸生长细胞提取物对异香草酸的去甲基化（反应I）需要钴胺素（I）进行还原激活，并被钴胺素（II）抑制和失活，这表明反应机制是处于还原态Co（I）的酶结合类咕啉对醚的甲基碳进行亲核攻击，而不是自由基攻击。只有苯甲醚被去甲基化；邻羟基或对羧基可提高去甲基化速率，但额外的间位取代基会降低去甲基化速率。异香草酸去甲基化速率为81nmol·min-1·（mg蛋白质）-1[0.76mM钴胺素（I）]，钴胺素（I）的表观动力学常数为：Km = 1.2mM，Vmax = 220nmol·min-1·（mg蛋白质）-1，Vmax/Km = 180nmol·min-1·（mg蛋白质）-1·mM-1。3,5-二羟基苯甲醚生长细胞提取物对3,5-二羟基苯甲醚的去甲基化（也是反应I）要慢得多。反应II不需要激活；甲基钴胺素（III）的比活性和特异性常数要低得多。
