Mutational analysis of carbamyl phosphate synthetase. Substitution of Glu841 leads to loss of functional coupling between the two catalytic domains of the synthetase subunit.
The synthetase subunit of Escherichia coli carbamyl phosphate synthetase has two catalytic nucleotide-binding domains, one involved in the activation of HCO3- and the second in phosphorylation of carbamate. Here we show that a Glu841----Lys841 substitution in a putative ATP-binding domain located in the carboxyl half of the synthetase abolishes overall synthesis of carbamyl phosphate with either glutamine or NH3 as the nitrogen source. Measurements of partial activities indicate that while HCO3(-)-dependent ATP hydrolysis at saturating concentrations of substrate proceeds at higher than normal rates, ATP synthesis from ADP and carbamyl phosphate is nearly completely suppressed by the mutation. These results indicate Glu841 to be an essential residue for the phosphorylation of carbamate in the terminal step of the catalytic mechanism. The Lys841 substitution also affects the kinetic properties of the HCO3- activation site. Both kcat and Km for ATP increase 10-fold, while Km for HCO3- is increased 100-fold. Significantly, NH3 decreases rather than stimulates Pi release from ATP in the HCO3(-)-dependent ATPase reaction. The increase in kcat of the HCO3(-)-dependent ATPase reaction, and an impaired ability of the Lys841 enzyme to catalyze the reaction of NH3 with carboxy phosphate, strongly argues for interactions between the two catalytic ATP sites that couple the formation of enzyme-bound carbamate with its phosphorylation.
Guillou
F
F
Department of Molecular Genetics, Public Health Research Institute, New York, New York 10016.
Liao
M
M
Garcia-Espana
A
A
Lusty
C J
CJ
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001639:Bicarbonates / Q000494:pharmacology; D001665:Binding Sites; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000737:chemistry* / Q000235:genetics; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry* / Q000235:genetics; D002221:Carbamyl Phosphate / Q000378:metabolism; D002384:Catalysis; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D001639:Bicarbonates; D002221:Carbamyl Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)