The Allosteric and Chemical Mechanism of Methylglyoxal Synthase

酶活性的变构调节是细胞对环境波动作出反应的最直接机制之一。虽然许多酶的变构性质和机制已经得到了非常详细的研究，但对这些系统的溶液动力学知之甚少。大肠杆菌甲基乙二醛合成酶是一种变构调节的同六聚体酶，将用于了解蛋白质动力学在多亚基酶的变构调节中的作用。1H-15N主酰胺核磁共振将使用TROSY脉冲序列技术进行分配，每个共振的动力学将在皮秒到毫秒的时间尺度上进行测量，包括t态和r态。等温微量热法将用于确定与构象变化相关的热量，并独立确定两个变构构象之间是否存在预先存在的平衡。在一个类似于三磷酸异构酶催化的反应中，MGS通过烯二酚中间体催化磷酸二羟丙酮的磷酸消除，形成甲基乙二醛的烯醇。质子从氧转移到氧的机制和磷酸化消除的机制将通过位点定向诱变、机制酶学、计算分析和x射线晶体学的结合来研究。磷酸化极化对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成酶复合物的结构和底物二羟丙酮磷酸的相应硫类似物的动力学来确定。甲基乙二醛合成酶和三磷酸酯异构酶的催化表面将进行比较，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.