Jay M, De Luca V, Ibrahim R K
Eur J Biochem. 1985 Dec 2;153(2):321-5. doi: 10.1111/j.1432-1033.1985.tb09304.x.
A novel O-methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to the 8-hydroxyl group of flavonols was purified about 1200-fold from Lotus flower buds, by precipitation with ammonium sulfate and successive chromatography on columns of Sephadex G-100, S-adenosyl-L-homocysteine--Agarose, hydroxyapatite and Polybuffer ion exchanger. The enzyme exhibited strict specificity for position 8 of 8-hydroxyquercetin and 8-hydroxykaempferol, a pH optimum at 7.9, a pI value of 5.5, an Mr of 55 X 10(3) and required Mg2+ and SH groups for activity. The Km values for 8-hydroxykaempferol and S-adenosyl-L-methionine were 1.3 microM and 53 microM, respectively. The data obtained from substrate interaction and product inhibition studies are expected for a steady-state ordered bi-bi mechanism, with 8-hydroxyflavonol binding before S-adenosyl-L-methionine followed by the release of S-adenosyl-L-homocysteine and 8-methoxyflavonol. An alternative mechanism that may also fit the data is the mono-iso Theorell-Chance with the inverse binding sequence and an isomerization step of the free enzyme.
一种新型的O-甲基转移酶可催化S-腺苷-L-甲硫氨酸的甲基转移至黄酮醇的8-羟基上，通过硫酸铵沉淀以及在Sephadex G-100柱、S-腺苷-L-高半胱氨酸-琼脂糖柱、羟基磷灰石柱和聚缓冲离子交换柱上的连续层析，从莲花花蕾中纯化得到该酶，纯化倍数约为1200倍。该酶对8-羟基槲皮素和8-羟基山奈酚的8位具有严格的特异性，最适pH为7.9，pI值为5.5，Mr为55×10³，且活性需要Mg²⁺和巯基。8-羟基山奈酚和S-腺苷-L-甲硫氨酸的Km值分别为1.3μM和53μM。从底物相互作用和产物抑制研究获得的数据符合稳态有序双底物双产物机制，即8-羟基黄酮醇在S-腺苷-L-甲硫氨酸之前结合，随后释放S-腺苷-L-高半胱氨酸和8-甲氧基黄酮醇。另一种可能符合这些数据的机制是具有反向结合顺序和游离酶异构化步骤的单异Theorell-Chance机制。
