Recombinant Mouse Muscle Adenylosuccinate Synthetase

Abstract

Vertebrates possess two isozymes of adenylosuccinate synthetase. The acidic isozyme is similar to the synthetase from bacteria and plants, being involved in the de novo biosynthesis of AMP, whereas the basic isozyme participates in the purine nucleotide cycle. Reported here is the first instance of overexpression and crystal structure determination of a basic isozyme of adenylosuccinate 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 M, 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 conformation of loops, involved in substrate recognition and stabilization of the transition 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 active site. Observed differences in ligand recognition among adenylosuccinate synthetases may be due in part to conformational variations in the IMP pocket of the ligand-free enzymes.

Variations in the Response of Mouse Isozymes of Adenylosuccinate Synthetase to Inhibitors of Physiological Relevance

“…AdSS2 is stable with respect to freeze/thaw cycles in buffers supplemented with 30% glycerol. Full-length AdSS1, with or without their N-terminal polyhistidyl tags, have identical kinetic parameters and crystallize under the same conditions (28). Similarly, truncation of the N-terminal polyhistidyl tag from AdSS2 does not change its kinetic properties (data not shown).…”

“…Mouse recombinant AdSS1 has a significantly lower K m for IMP than that reported for the basic isozyme isolated from either rat or rabbit (28). Reported K m values for adenylosuccinate synthetases vary considerably (2,6,28) due, in part, to variations in assay protocols and conditions of assay, as well as intrinsic differences in the synthetases themselves (2,6).…”

“…Construction of Full-length and Truncated Synthetases-The cloning of full-length AdSS1 into the expression plasmid pET28b was described previously (28). cDNA for mouse acidic adenylosuccinate synthetase (AdSS2) was kindly provided by Dr. F. B. Rudolph (Department of Biochemistry and Cell Biology, Rice University, Houston, TX) as a pSPORT1clone (11).…”

“…Sequence Alignments-DNA and protein sequences were aligned using Multalign (35 Expression and Purification of Recombinant Isozymes-AdSS1 was produced and purified as described previously (28). The same procedure was used for AdSS1-Tr.…”

“…Reported K m values for adenylosuccinate synthetases vary considerably (2,6,28) due, in part, to variations in assay protocols and conditions of assay, as well as intrinsic differences in the synthetases themselves (2,6). The low natural abundance of AdSS2 has been an impediment to its purification and rigorous evaluation (7,9,18).…”