5-Hydroxy-<em>N</em>-methylpyroglutamate Synthetase: EVIDENCE FOR AN α-KETOGLUTARYL ENZYME INTERMEDIATE FROM PARTITIONING STUDIES

The enzyme 5-hydroxy--methylpyroglutamate synthetase catalyzes the synthesis and hydrolysis of δ-amides of α-ketoglutarate. Previous kinetic studies suggested that these reactions proceed either by a two-step mechanism involving an α-ketoglutaryl enzyme intermediate, or by a mechanism involving direct nucleophilic attack in which an isomerization of the enzyme is rate-determining. To distinguish between these two mechanisms, we have measured the partitioning of the acyl group derived from α-ketoglutaramate and δ-ethyl, δ-propyl, and δ-benzyl α-ketoglutarate between water and an amine. Identical partitioning of the above four substrates between water and methylamine, water and ethanolamine, and water and aniline (esters only) suggests that the two-step mechanism is the correct one.

5 - 羟基 - γ - 甲基焦谷氨酸合成酶催化α - 酮戊二酸的δ - 酰胺的合成与水解。先前的动力学研究表明，这些反应要么通过涉及α - 酮戊二酸酶中间体的两步机制进行，要么通过一种涉及直接亲核攻击且酶的异构化是决速步骤的机制进行。为了区分这两种机制，我们测量了来自α - 酮戊二酸酰胺以及δ - 乙基、δ - 丙基和δ - 苄基α - 酮戊二酸的酰基在水和一种胺之间的分配。上述四种底物在水和甲胺、水和乙醇胺以及水和苯胺（仅酯类）之间相同的分配情况表明两步机制是正确的。