Conversion of non-allosteric methylglyoxal synthase into a homotropic allosteric enzyme by C-terminal deletion

Our previous study revealed that the Hill coefficient of Escherichia coli methylglyoxal synthase (EMGS) is higher than what we have calculated for MGS from Thermus sp. GH5 (TMGS). Amino acid sequence alignment of EMGS and TMGS shows that key residues of allosteric pathways in EMGS exist in TMGS as well, except Arg150 which plays a crucial role in forming a salt bridge with Asp20 in the neighboring subunit and consequently transfers the allosteric signal between the subunits. To equalize allosteric pathway in EMGS with TMGS, ten amino acid residues, containing Arg150, are omitted from the EMGS C-terminal tail. The resulting recombinant enzyme (EMGS-ΔC) surprisingly shows homotropic cooperative behavior in presence of dihydroxyacetone phosphate. Structural studies and irreversible thermoinactivation data shows EMGS-ΔC is not only more flexible but also less stable compared to wild-type EMGS. These data suggest EMGS C-terminal tail may play an important role in allosteric behavior and stability of wild-type EMGS and thus indicating that the homotropic cooperativity is arisen by binding of the substrate which pushes the pre-existing equilibrium between the relatively inactive (RI) and relatively active (RA) conformations.