Evidence that the catalytic and regulatory functions of carbamylphosphate synthetase from Escherichia coli are not dependent on oligomer formation.
Carbamyl-phosphate synthetase from Escherichia coli is an allosteric enzyme which undergoes reversible association reactions in phosphate buffer. The positive allosteric effectors, ornithine, inosine 5'-monophosphate (IMP), and ammonia, facilitate oligomer formation, whereas uridine 5'-monophosphate (UMP), a negative effector, prevents or decreases oligomer formation. When the enzyme is immobilized by reaction with activated Sepharose, under conditions where the enzyme exists only as a monomer, nearly full catalytic activity is retained and the effects of ornithine, IMP, and UMP on the catalytic activity as a function of MgATP concentration are not significantly altered. Gel-filtration chromatography on Sephadex G-200 of catalytic quantities of the enzyme in the presence of all substrates showed that the elution volume was the same as that measured for the enzyme under conditions where it is known to exist in the monomer form. The specific activity of the enzyme does not increase when the concentration of the enzyme is increased 100-fold from a concentration at which the enzyme exists as monomer to a level at which the enzyme exists predominantly as oligomer. These results indicate that the monomer form of the enzyme is the principle active species and that oligomer formation is not directly related to enzyme activity or enzyme regulation.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000495:Allosteric Site; D000641:Ammonia / Q000494:pharmacology; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007292:Inosine Nucleotides / Q000494:pharmacology; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D009952:Ornithine / Q000494:pharmacology; D010770:Phosphotransferases / Q000378:metabolism; D011485:Protein Binding
D007292:Inosine Nucleotides; D046911:Macromolecular Substances; D000641:Ammonia; D000255:Adenosine Triphosphate; D009952:Ornithine; D010770:Phosphotransferases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D008274:Magnesium