Carbamoylphosphate synthetase from Pseudomonas aeruginosa. Subunit composition, kinetic analysis and regulation.
Carbamoylphosphate synthetase from Pseudomonas aeruginosa is subject to repression by pyrimidines and significant derepression by limitation of arginine or pyrimidines. Carbamoylphosphate synthetase was purified to homogeneity from a derepressed strain of P. aeruginosa. The molecular weight of the enzyme was estimated to be 168 000 by sucrose gradient ultracentrifugation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme is composed of two non-identical subunits with molecular weights of 122 000 and 44 000. Cross-linking the enzyme prior to electrophoresis yielded an additional band corresponding to a molecular weight of 165 000, showing that the enzyme is composed of one of each subunit. The enzyme utilized either glutamine (Km 0.15 mM) or NH3 (Km 17 mM) and requires free Mg2+ for maximal activity with the optimal level between 4 mM and 10 mM. Hill plots of MgATP saturation data yielded coefficients of 1.2 and 1.4 at pH 8.0 and 8.5, respectively. A Hill equation was derived on the assumptions that MgATP binds at the same time to two distinct sub-sites as was shown to be the case for carbamoylphosphate synthetase from Escherichia coli and that these sub-sites are strictly non-interacting. The resulting theoretical Hill coefficients correspond very closely to the experimental coefficients. Carbamoylphosphate synthetase activity was subject to activation by ornithine and N-acetylornithine and feedback inhibition by UMP. Carbamoylphosphate synthetase from P. aeruginosa does not associate under all conditions examined, establishing that self-association does not play a role in regulation of enzyme activity as suggested by other workers for the enzyme from E. coli.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001426:Bacterial Proteins / Q000302:isolation & purification*; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D007700:Kinetics; D008025:Ligases / Q000302:isolation & purification*; D008274:Magnesium / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D008274:Magnesium