Studies on the structure-function relationships and regulation mechanisms of Escherichia coli adenylosuccinate synthetase

Molecular biological, biochemical, and biophysical techniques were utilized to explore structure-function relationship of Escherichia coli adenylosuccinate synthetase. For the first time, residues Arg303, Arg304 and Arg305 were implicated as the determinants in the recognition and binding of substrate L-aspartate. Replacing Arg305 with leucine resulted in over a 100-fold decrease in kcat, suggesting a possible role of Arg305 in catalysis. pH dependent kinetic studies indicated that the side chain of Gln224 is not only involved in recognition of IMP, but also in stabiling the intermediate 6-phosphoryl-IMP. Substitution of the side chain amide with a carboxylate group resulted in loss of catalytic activity. The enzymes with mutations on the interface region, R143K, R1431, and D231A, along with wild type AMPSase, were analyzed using MALDI mass spectroscopy, sedimentation equilibrium, and initial rate kinetics. Ligand-induced dimerization is suggested as the regulatory mechanism for AMPSase, with the monomer as the inactive or lower active species, with the dimer as the fully active form;The purA gene was subcloned into the prokaryotic expression vector pTrc99A for better expression. The new construct pTrpA produced a 10- to 20-fold increase in enzyme yield compared to the older expression vector PMS204