Investigating the role of the salt bridige between Asp100 and Arg80 in TMGS allosteric Behavior

The allosterically regulated  enzyme methylglyoxal synthase (MGS, EC 4.2.3.3)  is a homohexameric enzyme that catalyzes the conversion of dihydroxyacetone  phosphate (DHAP) to methylglyoxal and phosphate in the first step of the methylglyoxal bypass of glycolysis pathway. Thermophilic type of MGS was  first isolated and expressed from Thermus sp.GH5 (TMGS). Phosphate was found to be  a strong inhibitor and negative allosteric effector of the enzyme. According to the crystallographic structure of phosphate bound MGS, conformational changes in the conserved regiones of the protein closes the entry of the channel leading to the active site and affects the interresidual interaction at the monomer-monomer interface. New interaction between residues Arg80 and Asp100 occur upon phosphate binding to the enzyme and may provide a pathway by which allosteric inhibitory signal is transmitted through the intersubunit interface. To test this hypothesis, this two residues were mutated with Quik change site directed mutagenesis and allosteric behavior of mutated variants were investigated.