Liang P H, Lewis J, Anderson K S, Kohen A, D'Souza F W, Benenson Y, Baasov T
Department of Chemistry, Technion-Israel Institute of Technology, Haifa, Israel.
Biochemistry. 1998 Nov 17;37(46):16390-9. doi: 10.1021/bi981374w.
The mechanistic pathway for the reaction catalyzed by Kdo8P synthase has been investigated, and the cyclic bisphosphate 2 has been examined as a putative reaction intermediate. Two parallel approaches were used: (1) chemical synthesis of 2 and evaluation as an alternate substrate for the enzyme and (2) transient kinetic studies using rapid chemical quench methodology to provide direct observation and characterization of putative intermediate(s) during enzyme catalysis. The putative cyclic bisphosphate intermediate 2, possessing the stereochemistry of the beta-pyranose form, was synthesized and evaluated as a substrate and as an inhibitor of Kdo8P synthase. The substrate activity was examined by monitoring the release of anomeric phosphate over time using proton-decoupled 31P NMR spectroscopy. A very similar time course for the formation of inorganic phosphate was found in each experiment and the corresponding control experiment; i.e., no enzyme-catalyzed acceleration in the anomeric phosphate hydrolysis was detected. It was found however that 2 binds to the enzyme and is a competitive inhibitor with respect to phosphoenolpyruvate binding, having a Ki value of 35 microM. In a parallel study, we have performed single-turnover rapid chemical quench experiments to examine both the forward and reverse directions to identify a putative enzyme intermediate(s). Our results clearly demonstrate that the cyclic bisphosphate intermediate 2 does not accumulate under single-enzyme turnover conditions. This observation, coupled with the results obtained through the evaluation of synthetic 2 as a substrate, strongly suggests that the Kdo8P synthase catalytic pathway does not involve the formation of 2 as a reaction intermediate. Taken together, these combined results support the original hypothesis [Hedstrom, L., and Abeles, R. H. (1988) Biochem. Biophys. Res. Commun. 157, 816-820], which suggests a reaction pathway involving an acyclic bisphosphate intermediate 1.
已对Kdo8P合酶催化反应的机制途径进行了研究，并已将环状双磷酸酯2作为假定的反应中间体进行了考察。采用了两种平行的方法：（1）2的化学合成及其作为该酶替代底物的评估；（2）使用快速化学淬灭方法进行瞬态动力学研究，以直接观察和表征酶催化过程中的假定中间体。合成了具有β-吡喃糖形式立体化学的假定环状双磷酸酯中间体2，并将其作为底物和Kdo8P合酶的抑制剂进行评估。通过使用质子去耦31P NMR光谱监测异头磷酸随时间的释放来检测底物活性。在每个实验及其相应的对照实验中发现了非常相似的无机磷酸形成时间进程；即，未检测到酶催化的异头磷酸水解加速。然而，发现2与该酶结合，并且相对于磷酸烯醇丙酮酸结合是一种竞争性抑制剂，其Ki值为35μM。在一项平行研究中，我们进行了单周转快速化学淬灭实验，以研究正向和反向反应，以鉴定假定的酶中间体。我们的结果清楚地表明，环状双磷酸酯中间体2在单酶周转条件下不会积累。这一观察结果，结合通过将合成的2作为底物进行评估所获得的结果，强烈表明Kdo8P合酶催化途径不涉及形成2作为反应中间体。综上所述，这些综合结果支持了最初的假设[赫德斯特伦，L.，和阿贝莱斯，R. H.（1988年）生物化学与生物物理研究通讯157，816 - 820]，该假设提出了一种涉及无环双磷酸酯中间体1的反应途径。
