Carbamate kinase from Pseudomonas aeruginosa: purification, characterization, physiological role, and regulation.
Pseudomonas aeruginosa PAO1 possessed a carbamate kinase (CKase) distinct from carbamoylphosphate synthetase as well as from a constitutive acetate kinase which also catalyzes the phosphorylation of ADP by carbamoylphosphate. CKase was purified to homogeneity. Polyacrylamide gel electrophoresis of cross-linked CKase in the presence of sodium dodecyl sulfate showed that the enzyme consists of two subunits with identical molecular weights (37,000). The optimal pH of enzyme activity is 7.0. The double-reciprocal plot for carbamoylphosphate was linear at 2 mM ADP, yielding an apparent Km of 5 mM. However, at 0.25 mM ADP, the plot was concave upward, and a Hill plot of the data yielded a coefficient of 1.4. This apparent cooperativity at low ADP concentrations might serve to reduce the extent of catabolism of carbamoylphosphate under growth conditions yielding high energy charge. Experiments on the regulation of synthesis under various growth conditions showed a response to three regulatory signals: CKase was induced to high levels by anaerobiosis, induced to moderate levels by arginine, and repressed by ammonia. Thus, CKase expression is regulated in a manner that allows the enzyme to function as a provider of ammonia under aerobic conditions and of ATP under anaerobic conditions. ATP was an effective inhibitor of CKase activity; this inhibition provides the cell with an effective mechanism for avoiding a futile cycle resulting from the simultaneous operation of CKase and carbamoylphosphate synthetase when cells are grown in the presence of exogenous arginine.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000641:Ammonia / Q000494:pharmacology; D000693:Anaerobiosis; D001120:Arginine / Q000494:pharmacology; D002221:Carbamyl Phosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010755:Organophosphates / Q000378:metabolism; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D017851:Phosphotransferases (Carboxyl Group Acceptor); D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D010755:Organophosphates; C011632:acetyl phosphate; D002221:Carbamyl Phosphate; D000244:Adenosine Diphosphate; D000641:Ammonia; D000255:Adenosine Triphosphate; D001120:Arginine; D010770:Phosphotransferases; D017851:Phosphotransferases (Carboxyl Group Acceptor); C019288:carbamate kinase