The Allosteric and Chemical Mechanism of Methylglyoxal Synthase

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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.