Shoolingin-Jordan P M, Spencer P, Sarwar M, Erskine P E, Cheung K-M, Cooper J B, Norton E B
School of Biological Sciences, Southampton University, Bassett Crescent East, Southampton SO16 7PX, UK.
Biochem Soc Trans. 2002 Aug;30(4):584-90. doi: 10.1042/bst0300584.
5-Aminolaevulinic acid dehydratase catalyses the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid. The studies described highlight the importance of a bivalent metal ion and two active-site lysine residues for the functioning of 5-aminolaevulinic acid dehydratase. Dehydratases fall into two main categories: zinc-dependent enzymes and magnesium-dependent enzymes. Mutations that introduced zinc-binding ligands into a magnesium-dependent enzyme conferred an absolute requirement for zinc. Mutagenesis of lysine residues 247 and 195 in the Escherichia coli enzyme lead to dramatic effects on enzyme activity, with lysine 247 being absolutely essential. Mutation of either lysine 247 or 195 to cysteine, and treatment of the mutant enzyme with 2-bromethylamine, resulted in the recovery of substantial enzyme activity. The effects of the site-directed alkylating inhibitor, 5-chlorolaevulinic acid, and 4,7-dioxosebacic acid, a putative intermediate analogue, were investigated by X-ray crystallography. These inhibitors reacted with both active-site lysine residues. The role of these two lysine residues in the enzyme mechanism is discussed.
5-氨基乙酰丙酸脱水酶催化两分子5-氨基乙酰丙酸形成胆色素原。所述研究突出了二价金属离子和两个活性位点赖氨酸残基对5-氨基乙酰丙酸脱水酶功能的重要性。脱水酶主要分为两类：锌依赖性酶和镁依赖性酶。将锌结合配体引入镁依赖性酶的突变导致对锌的绝对需求。大肠杆菌酶中赖氨酸残基247和195的诱变对酶活性产生显著影响，其中赖氨酸247是绝对必需的。将赖氨酸247或195突变为半胱氨酸，并使用2-溴乙胺处理突变酶，可使酶活性得到大幅恢复。通过X射线晶体学研究了定点烷基化抑制剂5-氯乙酰丙酸和假定的中间类似物4,7-二氧代癸二酸的作用。这些抑制剂与两个活性位点赖氨酸残基均发生反应。讨论了这两个赖氨酸残基在酶作用机制中的作用。
