Department of Chemistry, Takatsuki, Osaka 569-8686, Japan.
Department of Biochemistry, Osaka Medical College, Daigamumachi 2-7, Takatsuki, Osaka 569-8686, Japan.
J Biochem. 2020 Apr 1;167(4):357-364. doi: 10.1093/jb/mvz100.
Threonine synthase catalyses the conversion of O-phospho-l-homoserine and a water molecule to l-threonine and has the most complex catalytic mechanism among the pyridoxal 5'-phosphate-dependent enzymes. In order to study the less-characterized earlier stage of the catalytic reaction, we studied the reaction of threonine synthase with 2-amino-5-phosphonopentanoate, which stops the catalytic reaction at the enamine intermediate. The global kinetic analysis of the triphasic spectral changes showed that, in addition to the theoretically expected pathway, the carbanion is rapidly reprotonated at Cα to form an aldimine distinct from the external aldimine directly formed from the Michaelis complex. The Kd for the binding of inhibitor to the enzyme decreased with increasing pH, showing that the 2-amino-group-unprotonated form of the ligand binds to the enzyme. On the other hand, the rate constants for the proton migration steps within the active site are independent of the solvent pH, indicating that protons are shared by the active dissociative groups and are not exchanged with the solvent during the course of catalysis. This gives an insight into the role of the phosphate group of the substrate, which may increase the basicity of the ε-amino group of the catalytic lysine residue in the active site.
苏氨酸合酶催化 O-磷酸-L-高丝氨酸和一个水分子转化为 L-苏氨酸，其催化机制是吡哆醛 5′-磷酸依赖酶中最复杂的。为了研究催化反应中特征较少的早期阶段，我们研究了苏氨酸合酶与 2-氨基-5-膦戊酸的反应，该反应在烯胺中间物处停止催化反应。三相光谱变化的整体动力学分析表明，除了理论上预期的途径外，碳负离子在 Cα 处迅速质子化，形成不同于直接从 Michaelis 复合物形成的外部亚胺的醛亚胺。抑制剂与酶结合的 Kd 值随 pH 值的增加而降低，表明配体的 2-氨基未质子化形式与酶结合。另一方面，活性位点内质子迁移步骤的速率常数与溶剂 pH 值无关，表明质子由活性解离基团共享，并且在催化过程中不与溶剂交换。这深入了解了底物磷酸基团的作用，其可能增加活性位点中催化赖氨酸残基的ε-氨基的碱性。
