Mechanism of carbamoyl phosphate synthetase from Escherichia coli

Abstract

The conflicting data on the binding of the two molecules of ATP that are involved in the overall reaction catalyzed by carbamoyl-phosphate synthetase (CPS) of Escherichia coli, and a mechanism recently proposed for this reaction, has led us to reexamine ATP binding using pulse/chase techniques. With [γ-32 P]ATP and bicarbonate in the pulse solution, there is a positive intercept at zero time of approximately 1 mol P i /mol CPS in the plot of 32 P i formation against time, irrespective of whether the incubation is terminated by the addition of acid or by addition of a chase solution containing glutamine, excess unlabeled ATP and bicarbonate. The intercept is decreased to about 50% if the excess unlabeled ATP is added prior to the addition of the glutamine. These are the expected results if the intercept reflects the reversible formation of enzyme-bound ADP and carboxyphosphate. Approximately 0.6 mol carbamoyl [ 32 P]phosphate/mol enzyme is formed in these experiments when the pulse step is terminated by addition to the chase solution. The ATP molecule that provides the phosphoryl group of carbamoyl phosphate, therefore, also binds to the enzyme in the absence of ammonia or glutamine and reacts in the chase to give carbamoyl phosphate before it can dissociate from the enzyme. At 1 mM ATP, the binding of both ATP molecules is essentially complete at 2.5 s, but the dissociation of the ATP that yields carbamoyl phosphate is extremely slow (t 1/2 of about 6 min at 22°C; HCO Ϫ 3 , 40 mM), although it is faster in the absence of bicarbonate. The extreme sequestration from the aqueous environment of this ATP allows the enzyme-ATP complex to be separated from the surrounding ATP by centrifugal gel filtration. After two successive steps of gel filtration through Sephadex G-50 equilibrated with unlabeled ATP and bicarbonate, the majority of the radioactivity remaining in the solution is bound to the enzyme and is released as [γ-32 P]ATP if acid is added, or is converted to carbamoyl [ 32 P]phosphate by addition to chase solution, without concomitant release of 32 P i . K ϩ is necessary in the pulse solution, but not in the chase solution, to demonstrate this binding. These findings and other confirmatory experiments demonstrate conclusively that, in the presence of K ϩ , both ATP molecules bind to the enzyme in the absence of ammonia or glutamine. The bound ATP that yields P i in the overall reaction is replaced relatively rapidly by exchange and by hydrolysis in the bicarbonate-dependent ATPase activity of the enzyme, whereas the bound ATP that provides the phosphoryl group of carbamoyl phosphate is replaced very slowly. The temporal pattern of carbamoyl [ 32 P]phosphate formation from [γ-32 P]ATP, in pulse/chase experiments in which a small concentration of ammonia is added to the pulse solution, shows that, in the normal enzyme reaction, this last ATP molecule binds to the enzyme before ammonia.These findings exclude a recently proposed mechanism [Kothe, M., Eroglu, B., Mazza, H., Samudera, H. & Powers- Lee, S. (1997) Pr...

Understanding Carbamoyl Phosphate Synthetase Deficiency: Impact of Clinical Mutations on Enzyme Functionality

“…The decreased accessibility of both phosphorylation sites is reflected in the increased K m values for the substrates of both partial reactions observed in this mutant (Table 3). Previous mechanistic studies with CPSI and E. coli CPS [45][46][47] agree with the concerted opening of both sites that is supported by the observations with the V640R mutant. In these studies ammonia (or glutamine, in the case of E. coli CPS) triggered rapid formation and dissociation of all the products from the enzyme complex containing ADP, carboxyphosphate and ATP.…”

Deciphering the Metabolic Pathway Difference Between Saccharopolyspora pogona and Saccharopolyspora spinosa by Comparative Proteomics and Metabonomics

“…xanthosine 5 -phosphate (XMP), carbamoyl-phosphate synthase (carA/carB) catalyzes the synthesis of carbamoyl-phosphate, which belongs to the rate-limiting enzyme of purine and pyrimidine nucleotide metabolism respectively (Rubio et al, 1998;Pimkin and Markham, 2009). The two enzymes as well as the relevant metabolites, such as xanthosine 5 -phosphate, L-glutamine and dihydroorotate, were all present at higher abundance in S. pogona, indicating that S. pogona has strong nucleotide metabolism ability.…”

The Carbamoyl-phosphate Synthetase of Pyrococcus furiosus Is Enzymologically and Structurally a Carbamate Kinase

“…Furthermore, our initial structural data obtained by x-ray crystallography clearly show that the pyrococcal enzyme exhibits a three-dimensional structure and quaternary organization that are highly similar to those of enterococcal CK and that are very different from those of CPS. Although the CK structure (11) reveals the existence of two catalytic sites/enzyme dimer, the relative orientation of the sites and the absence of intramolecular tunnels joining them exclude the possibility of catalytic collaboration between the two sites that is required for the synthesis of CP from bicarbonate and ammonia in three steps (bicarbonate phosphorylation, carbamate formation, and carbamate phosphorylation) that characterizes the mechanism of CPS (10,39). In summary, all indicate that except for its extreme thermostability and low activity at normal temperatures, 3 the pyrococcal enzyme is endowed with the characteristics of classical CKs.…”