Recombinant mouse muscle adenylosuccinate synthetase - Overexpression, kinetics, and crystal structure

Vertebrates possess two isozymes of adenylosuccinate synthetase. The acidic isozyme is similar to the synthetase from bacteria and plants, being invol ved in the de novo biosynthesis of AMP, whereas the basic isozyme participa tes in the purine nucleotide cycle. Reported here is the first instance of overexpression and crystal structure determination of a basic isozyme of ad enylosuccinate synthetase. The recombinant mouse muscle enzyme purified to homogeneity in milligram quantities exhibits a specific activity comparable with that of the rat muscle enzyme isolated from tissue and K-m parameters for GTP, IMP, and L-aspartate (12, 45, and 140 mum, respectively) similar to those of the enzyme from Escherichia coli. The mouse muscle and E. coli enzymes have similar polypeptide folds, differing primarily in the conforma tion of loops, involved in substrate recognition and stabilization of the t ransition state. Residues 65-68 of the muscle isozyme adopt a conformation not observed in any previous synthetase structure. In its new conformation, segment 65-68 forms intramolecular hydrogen bonds with residues essential for the recognition of IMP and, in fact, sterically excludes IMP from the a ctive site. Observed differences in ligand recognition among adenylosuccina te synthetases may be due in part to conformational variations in the IMP p ocket of the ligand-free enzymes.