Deconstruction of the catalytic array within the amidotransferase subunit of carbamoyl phosphate synthetase.
Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme consists of a large synthetase subunit, and a small amidotransferase subunit, which belongs to the Triad family of glutamine amidotransferases. Previous studies have established that the reaction mechanism of the small subunit proceeds through the formation of a gamma-glutamyl thioester with Cys-269. The roles in the hydrolysis of glutamine played by the conserved residues, Glu-355, Ser-47, Lys-202, and Gln-273, were determined by mutagenesis. In the X-ray crystal structure of the H353N mutant, Ser-47 and Gln-273 interact with the gamma-glutamyl thioester intermediate [Thoden, J. B., Miran, S. G., Phillips, J. C., Howard, A. J., Raushel, F. M., and Holden, H. M. (1998) Biochemistry 37, 8825-8831]. The mutants E355D and E355A have elevated values of K(m) for glutamine, but the overall carbamoyl phosphate synthesis reaction is unperturbed. E355Q does not significantly affect the bicarbonate-dependent ATPase or glutaminase partial reactions. However, this mutation almost completely uncouples the two partial reactions such that no carbamoyl phosphate is produced. The partial recovery of carbamoyl phosphate synthesis activity in the double mutant E355Q/K202M argues that the loss of activity in E355Q is at least partly due to additional interactions between Gln-355 and Lys-202 in E355Q. The mutants S47A and Q273A have elevated K(m) values for glutamine while the V(max) values are comparable to that of the wild-type enzyme. It is concluded that contrary to the original proposal for the catalytic triad, Glu-355 is not an essential residue for catalysis. The results are consistent with Ser-47 and Gln-273 playing significant roles in the binding of glutamine.
Huang
X
X
Department of Chemistry, Texas A&M University, College Station, Texas, 77843, USA.
Raushel
F M
FM
D000244:Adenosine Diphosphate / Q000737:chemistry; D000878:Anthranilate Synthase; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry* / Q000235:genetics; D002221:Carbamyl Phosphate / Q000737:chemistry; D020134:Catalytic Domain; D005972:Glutaminase / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019884:Nitrogenous Group Transferases / Q000737:chemistry*; D016133:Polymerase Chain Reaction
D005973:Glutamine; D002221:Carbamyl Phosphate; D000244:Adenosine Diphosphate; D019884:Nitrogenous Group Transferases; D005972:Glutaminase; D000878:Anthranilate Synthase; C020655:anthranilate synthase, glutamine amidotransferase subunit; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)