title: Regeneration of active enzyme by formation of hybrids from inactive derivatives: implications for active sites shared between polypeptide chains of aspartate transcarbamoylase.
abstract: Crystallographic studies of Escherichia coli aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) in conjunction with chemical modification experiments have led to the suggestion that the active sites of the enzyme are at the interfaces between adjacent polypeptide chains of the catalytic trimers and involve joint participation of amino acid residues from the adjoining chains. However, the precise locations of the active sites and of the residues involved in catalysis are not known. To test the hypothesis that the active sites are shared between chains, we constructed hybrid trimers in which two chains were modified at one presumed active site residue and the third chain was altered at a different active site residue. One parental trimer was a reduced pyridoxal phosphate derivative in which lysine-84 was modified and the other was a mutant protein in which tyrosine-165 was converted to serine by site-directed mutagenesis. Incubating mixtures of these two virtually inactive derivatives under conditions promoting interchain exchange led to a large increase in enzyme activity corresponding approximately to the formation of one active site per trimer. The purified hybrid trimers, containing either two pyridoxylated and one mutant chain or vice versa, had 23% and 28%, respectively, the activity of native wild-type catalytic trimers, compared to 5% and 3% for the parental trimers. The most likely explanation for this large increase in activity is the formation of one "native" active site in each of the hybrid trimers. The results constitute strong evidence for shared active sites in aspartate transcarbamoylase.
mesh_terms: D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001665:Binding Sites; D003461:Crystallography; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation
chemical_list: D046911:Macromolecular Substances; D001221:Aspartate Carbamoyltransferase