SUBSTRATE SYNERGISM AND THE STEADY-STATE KINETIC REACTION-MECHANISM FOR EPSP SYNTHASE FROM ESCHERICHIA-COLI
SUBSTRATE SYNERGISM AND THE STEADY-STATE KINETIC REACTION-MECHANISM FOR EPSP SYNTHASE FROM ESCHERICHIA-COLI
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DOI：
10.1021/bi00139a016
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发表时间：
1992-06-23
期刊：
影响因子：
2.9
通讯作者：
SIKORSKI, JA
中科院分区：
文献类型：
--
作者：
GRUYS, KJ;WALKER, MC;SIKORSKI, JA
大肠杆菌5-烯醇式乙酰莽草酸-3-磷酸合酶（EPSPS，EC 2.5.1.19）的先前研究表明，该酶在正向方向上的动力学反应机制是平衡有序的，首先结合莽草酸3-磷酸（S3 P），然后结合磷酸烯醇式丙酮酸（PEP）。然而，该实验室最近的结果表明，PEP和PEP类似物与游离EPSPS的直接结合表明酶具有更随机的特性。稳态动力学和光谱研究表明，E。coli EPSPS确实遵循随机动力学机制。与S3 P和PEP的初速度研究表明，PEP添加到一个正常的交叉模式的竞争性底物抑制。提出底物抑制通过PEP在S3 P位点的竞争性结合发生[K（i（PEP））= 6-8 mM]。为了测试生产性EPSPS.PEP二元复合物，用莽草酸和PEP作为底物评价EPSPS的反应级数。该反应的机制是平衡有序的，PEP结合首先给出PEP的K（ia）值，其与独立测量的0.39 mM的K（d）（莽草酸K（m）= 25 mM）一致。该研究的结果还表明，S3 P的3-磷酸部分相对于该位置的羟基提供8.7 kcal/mol的结合能。超过60%的这种结合能表示在底物与酶的结合中，而不是朝向增加k（cat）。与正常反应相比，草甘膦对莽草酸周转的抑制作用较差，结合能力损失约为8 × 10（4），这与EPSPS.草甘膦二元复合物的独立测量K（d）为12 mM一致。然而，EPSPS.草甘膦复合物诱导莽草酸结合比EPSPS. PEP大7倍。发现磷酸羧基丙二烯酯和（Z）-3-氟-PEP是该酶的强抑制剂，其对除了PEP位点之外的S3 P结合结构域具有令人惊讶的亲和力，如动力学和通过用P-31 NMR直接观察所测量的。收集的数据表明，真正的动力学机制EPSPS在正向方向是随机的与底物和抑制剂之间发生的协同结合。协同作用解释了该机制如何与S3 P和PEP随机，但对于莽草酸周转，PEP首先结合的平衡有序。协同作用也解释了草甘膦如何成为正常反应的强抑制剂，但与莽草酸周转相比效果较差。
Previous studies of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) have suggested that the kinetic reaction mechanism for this enzyme in the forward direction is equilibrium ordered with shikimate 3-phosphate (S3P) binding first followed by phosphoenolpyruvate (PEP). Recent results from this laboratory, however, measuring direct binding of PEP and PEP analogues to free EPSPS suggest more random character to the enzyme. Steady-state kinetic and spectroscopic studies presented here indicate that E. coli EPSPS does indeed follow a random kinetic mechanism. Initial velocity studies with S3P and PEP show competitive substrate inhibition by PEP added to a normal intersecting pattern. Substrate inhibition is proposed to occur by competitive binding of PEP at the S3P site [K(i(PEP)) = 6-8 mM]. To test for a productive EPSPS.PEP binary complex, the reaction order of EPSPS was evaluated with shikimic acid and PEP as substrates. The mechanism for this reaction is equilibrium ordered with PEP binding first giving a K(ia) value for PEP in agreement with the independently measured K(d) of 0.39 mM (shikimate K(m) = 25 mM). Results from this study also show that the 3-phosphate moiety of S3P offers 8.7 kcal/mol in binding energy versus a hydroxyl in this position. Over 60% of this binding energy is expressed in binding of substrate to enzyme rather than toward increasing k(cat). Glyphosate inhibition of shikimate turnover was poor with approximately 8 X 10(4) loss in binding capacity compared to the normal reaction, consistent with the independently measured K(d) of 12 mM for the EPSPS.glyphosate binary complex. The EPSPS.glyphosate complex induces shikimate binding, however, by a factor of 7 greater than EPSPS.PEP. Carboxyallenyl phosphate and (Z)-3-fluoro-PEP were found to be strong inhibitors of the enzyme that have surprising affinity for the S3P binding domain in addition to the PEP site as measured both kinetically and by direct observation with P-31 NMR. The collective data indicate that the true kinetic mechanism for EPSPS in the forward direction is random with synergistic binding occurring between substrates and inhibitors. The synergism explains how the mechanism can be random with S3P and PEP, but yet equilibrium ordered with PEP binding first for shikimate turnover. Synergism also accounts for how glyphosate can be a strong inhibitor of the normal reaction, but poor versus shikimate turnover.