Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.
Biochemistry (Mosc). 2023 May;88(5):600-609. doi: 10.1134/S0006297923050036.
O-acetylhomoserine sulfhydrylase is one of the key enzymes in biosynthesis of methionine in Clostridioides difficile. The mechanism of γ-substitution reaction of O-acetyl-L-homoserine catalyzed by this enzyme is the least studied among the pyridoxal-5'-phosphate-dependent enzymes involved in metabolism of cysteine and methionine. To clarify the role of active site residues Tyr52 and Tyr107, four mutant forms of the enzyme with replacements of these residues with phenylalanine and alanine were generated. Catalytic and spectral properties of the mutant forms were investigated. The rate of γ-substitution reaction catalyzed by the mutant forms with replaced Tyr52 residue decreased by more than three orders of magnitude compared to the wild-type enzyme. The Tyr107Phe and Tyr107Ala mutant forms practically did not catalyze this reaction. Replacements of the Tyr52 and Tyr107 residues led to the decrease in affinity of apoenzyme to coenzyme by three orders of magnitude and changes in the ionic state of the internal aldimine of the enzyme. The obtained results allowed us to assume that Tyr52 is involved in ensuring optimal position of the catalytic coenzyme-binding lysine residue at the stages of C-α-proton elimination and elimination of the side group of the substrate. Tyr107 could act as a general acid catalyst at the stage of acetate elimination.
O-乙酰高丝氨酸硫内酯酶是艰难梭菌中蛋氨酸生物合成的关键酶之一。在涉及半胱氨酸和蛋氨酸代谢的依赖于吡哆醛-5'-磷酸的酶中，该酶催化的 O-乙酰-L-高丝氨酸的γ-取代反应的机制研究最少。为了阐明活性位点残基 Tyr52 和 Tyr107 的作用，生成了四个突变体形式的酶，这些残基被苯丙氨酸和丙氨酸取代。研究了突变体形式的催化和光谱特性。与野生型酶相比，催化γ-取代反应的突变体形式 Tyr52 取代残基的速率降低了三个数量级以上。Tyr107Phe 和 Tyr107Ala 突变体形式实际上几乎不催化该反应。Tyr52 和 Tyr107 残基的取代导致apoenzyme 与辅酶的亲和力降低了三个数量级，并改变了酶内部亚胺的离子状态。获得的结果使我们假设 Tyr52 参与确保催化辅酶结合赖氨酸残基在 C-α-质子消除和底物侧基消除阶段的最佳位置。Tyr107 可以在乙酸盐消除阶段充当通用酸催化剂。
