Center for Computational Sciences, University of Tsukuba , Tennodai 1-1-1, Tsukuba 305-8577, Japan.
J Am Chem Soc. 2014 Mar 26;136(12):4525-33. doi: 10.1021/ja408780c. Epub 2014 Mar 13.
Threonine synthase catalyzes the most complex reaction among the pyridoxal-5'-phosphate (PLP)-dependent enzymes. The important step is the addition of a water molecule to the Cβ-Cα double bond of the PLP-α-aminocrotonate aldimine intermediate. Transaldimination of this intermediate with Lys61 as a side reaction to form α-ketobutyrate competes with the normal addition reaction. We previously found that the phosphate ion released from the O-phospho-l-homoserine substrate plays a critical role in specifically promoting the normal reaction. In order to elucidate the detailed mechanism of this "product-assisted catalysis", we performed comparative QM/MM calculations with an exhaustive search for the lowest-energy-barrier reaction pathways starting from PLP-α-aminocrotonate aldimine intermediate. Satisfactory agreements with the experiment were obtained for the free energy profile and the UV/vis spectra when the PLP pyridine N1 was unprotonated and the phosphate ion was monoprotonated. Contrary to an earlier proposal, the base that abstracts a proton from the attacking water was the ε-amino group of Lys61 rather than the phosphate ion. Nevertheless, the phosphate ion is important for stabilizing the transition state of the normal transaldimination to form l-threonine by making a hydrogen bond with the hydroxy group of the l-threonine moiety. The absence of this interaction may account for the higher energy barrier of the side reaction, and explains the mechanism of the reaction specificity afforded by the phosphate ion product. Additionally, a new mechanism, in which a proton temporarily resides at the phenolate O3' of PLP, was proposed for the transaldimination process, a prerequisite step for the catalysis of all the PLP enzymes.
苏氨酸合酶催化吡哆醛-5'-磷酸（PLP）依赖性酶中最复杂的反应。重要的步骤是在 PLP-α-氨基巴豆酸亚胺中间物的 Cβ-Cα双键上加一个水分子。该中间物与赖氨酸 61 侧反应发生 transaldimination 形成α-酮丁酸，与正常加成反应竞争。我们之前发现，从 O-磷酸-l-高丝氨酸底物释放的磷酸离子在特异性促进正常反应中起着关键作用。为了阐明这种“产物辅助催化”的详细机制，我们进行了比较 QM/MM 计算，对从 PLP-α-氨基巴豆酸亚胺中间物开始的最低能垒反应途径进行了详尽的搜索。当 PLP 吡啶 N1 未质子化且磷酸离子单质子化时，自由能曲线和 UV/vis 光谱与实验结果吻合良好。与早期的提议相反，从进攻水中提取质子的碱是赖氨酸 61 的ε-氨基，而不是磷酸离子。然而，磷酸离子对于通过与 l-苏氨酸部分的羟基形成氢键来稳定正常 transaldimination 形成 l-苏氨酸的过渡态很重要。这种相互作用的缺失可能导致侧反应的能垒更高，并解释了磷酸离子产物赋予反应特异性的机制。此外，还提出了一种新的机制，其中质子暂时驻留在 PLP 的苯氧基 O3'，这是所有 PLP 酶催化的先决步骤。
