The Allosteric and Chemical Mechanism of Methylglyoxal Synthase

酶活性的变构调节是细胞响应环境波动的最直接机制之一。 虽然已经非常详细地研究了许多酶的变构性质和机制，但对这些系统的溶液动力学知之甚少。 E.大肠杆菌甲基乙二醛合酶，变构调节的同六聚体酶，将被用来了解蛋白质动力学的作用，在变构调节的多亚基酶。 将使用TROSY脉冲序列技术分配1H-15 N骨架酰胺NMR共振，并在T状态和R状态下以皮秒至毫秒的时间尺度测量每个共振的动力学。 将使用等温微量热法测定与构象变化相关的热量，并独立测定两种变构构象之间是否存在预先存在的平衡。 在一个类似于磷酸丙糖异构酶催化的反应中，MGS催化从磷酸二羟丙酮中消除磷酸盐，通过烯二醇中间体形成甲基乙二醛的烯醇。 质子从氧转移到氧的机制和磷酰基消除的机制将通过定点诱变、机械酶学、计算分析和X射线晶体学的组合来研究。 磷酰基极化催化的程度和重要性将通过检查已知抑制剂的新型硫类似物的甲基乙二醛合酶复合物的结构和底物磷酸二羟丙酮的相应硫类似物的动力学来确定。 将比较丙酮醛合酶和磷酸丙糖异构酶的催化表面，以了解每种酶如何控制其相应的反应途径。

The allosteric regulation of enzyme activity is one of the most direct mechanisms for a cell to respond to fluctuations in its environment. While the allosteric properties and mechanism of a number of enzymes have been studied in great detail, there is relatively little known about the solution dynamics of these systems. E. coli methylglyoxal synthase, an allosterically regulated homohexameric enzyme, will be used to understand the role of protein dynamics in the allosteric regulation of a multisubunit enzyme. The 1H-15N backbone amide NMR resonances will be assigned using TROSY pulse-sequence technology, and the dynamics of each resonance will be measured on the picosecond to millisecond timescale in both the T-state and the R-state. Isothermal microcalorimetry will be used to determine the heat associated with the conformational change and to independently determine if a pre-existing equilibrium exists between the two allosteric conformations. In a reaction reminiscent of that catalyzed by triosephosphate isomerase, MGS catalyzes the elimination of phosphate from dihydroxyacetone phosphate to form the enol of methylglyoxal via an enediolic intermediate. Both the mechanism of proton transfer from oxygen to oxygen and the mechanism of phosphoryl elimination will be studied by a combination of site directed mutagenesis, mechanistic enzymology, computational analyses, and X-ray crystallography. The extent and importance of phosphoryl polarization to catalysis will be determined both by examining the structure of methylglyoxal synthase complexes of novel sulfur analogues of known inhibitors and by the kinetics of corresponding sulfur analogues of the substrate, dihydroxyacetone phosphate. The catalytic surfaces of methylglyoxal synthase and triosephosphate isomerase will be compared to understand how each enzyme controls its corresponding reaction pathway.