Kohen A, Jakob A, Baasov T
Department of Chemistry, Technion-Israel Institute of Technology, Haifa.
Eur J Biochem. 1992 Sep 1;208(2):443-9. doi: 10.1111/j.1432-1033.1992.tb17206.x.
The anomeric specificity and the steady-state kinetic mechanism of homogeneous 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase were investigated. The open-chain 4-deoxy analogue of arabinose-5-phosphate (Ara5P), which is structurally prohibited from undergoing ring closure, was synthesized and tested as a substrate for the synthase. It was found that the analogue functions as a substrate with a similar kcat value to that of the original substrate. The kcat/Km value for the natural substrate is seven-times greater than that of the 4-deoxy analogue. However, taking into account the 9.5% and approximately 1% concentrations of the aldehyde forms of the 4-deoxy analogue and Ara5P in solution, then the 'true' Km values must be in the range 31.5 microM and 0.26 microM, respectively, requiring about a 3 kcal/mol contribution to the binding energy by the 4-hydroxyl group of Ara5P. The data provides evidence that the enzyme acts upon the acyclic form of the natural substrate. The steady-state kinetic study of KDO8P synthase was analyzed via inhibition using the products KDO8P and inorganic phosphate, and D-ribose-5-phosphate as a dead-end inhibitor. First, intersecting lines in double-reciprocal plots of initial-velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism for the enzyme-catalyzed reaction. The inhibition by D-ribose-5-phosphate is competitive for Ara5P and uncompetitive for phosphoenolpyruvate (P-pyruvate). These inhibition patterns are consistent with the model wherein P-pyruvate binding precedes that of Ara5P binding. Furthermore, this order of substrate binding was supported by the observations that KDO8P is a competitive inhibitor for P-pyruvate binding, supporting the concept that KDO8P and P-pyruvate bind to the same enzyme form, and noncompetitively with respect to Ara5P. In addition, the inhibition by inorganic phosphate is noncompetitive with respect to both P-pyruvate and Ara5P, suggesting an apparent ordered release of products such that Pi first, followed by KDO8P. In conclusion, these data suggest a steady-state kinetic mechanism for KDO8P synthase where P-pyruvate binding precedes that of Ara5P, followed by the ordered release of inorganic phosphate and KDO8P.
研究了均一的3-脱氧-D-甘露-2-辛酮糖酸-8-磷酸（KDO8P）合酶的异头特异性和稳态动力学机制。合成了阿拉伯糖-5-磷酸（Ara5P）的开链4-脱氧类似物，其在结构上无法进行环化，并将其作为该合酶的底物进行测试。发现该类似物作为底物发挥作用，其催化常数（kcat）值与原始底物相似。天然底物的kcat/Km值比4-脱氧类似物大7倍。然而，考虑到溶液中4-脱氧类似物和Ara5P醛形式的浓度分别为9.5%和约1%，那么“真实”的Km值必须分别在31.5微摩尔和0.26微摩尔范围内，这需要Ara5P的4-羟基对结合能贡献约3千卡/摩尔。这些数据提供了证据，表明该酶作用于天然底物的无环形式。通过使用产物KDO8P和无机磷酸盐以及D-核糖-5-磷酸作为终产物抑制剂进行抑制作用，对KDO8P合酶进行了稳态动力学研究。首先，如果底物浓度在微摩尔范围内，初始速度数据的双倒数图中的相交线表明该酶催化反应为有序机制。D-核糖-5-磷酸的抑制作用对Ara5P是竞争性的，对磷酸烯醇丙酮酸（P-丙酮酸）是非竞争性 的。这些抑制模式与P-丙酮酸结合先于Ara5P结合的模型一致。此外，底物结合的这种顺序得到了以下观察结果的支持：KDO8P是P-丙酮酸结合的竞争性抑制剂，支持了KDO8P和P-丙酮酸与相同酶形式结合且对Ara5P是非竞争性的概念。此外，无机磷酸盐的抑制作用对P-丙酮酸和Ara5P都是非竞争性的，这表明产物明显按顺序释放，即首先是磷酸根离子（Pi），然后是KDO8P。总之，这些数据表明KDO8P合酶的稳态动力学机制是P-丙酮酸结合先于Ara5P，随后是无机磷酸盐和KDO8P的有序释放。
