Role of the four conserved histidine residues in the amidotransferase domain of carbamoyl phosphate synthetase.
Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from ATP, bicarbonate, and glutamine. The amidotransferase activity of this enzyme is catalyzed by the smaller of the two subunits of the heterodimeric protein. The roles of four conserved histidine residues within this subunit were probed by site-directed mutagenesis to asparagine. The catalytic activities of the H272N and H341N mutants are not significantly different than that of the wild-type enzyme. The H353N mutant is unable to utilize glutamine as a nitrogen source in the synthetase reaction or the partial glutaminase reaction. However, binding to the glutamine active site is not impaired in the H353N enzyme since glutamine is found to activate the partial ATPase reaction by 40% with a Kd of 54 microM. The H312N mutant has a Michaelis constant for glutamine that is 2 orders of magnitude larger than the wild-type value, but the maximal rate of glutamine hydrolysis is unchanged. These results are consistent with His-353 functioning as a general acid/base catalyst for proton transfers while His-312 serves a critical role for the binding of glutamine to the active site.
Miran
S G
SG
Department of Chemistry, Texas A&M University, College Station 77843.
Chang
S H
SH
Raushel
F M
FM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005972:Glutaminase / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000031:analogs & derivatives / Q000494:pharmacology; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid
D015345:Oligonucleotide Probes; D005973:Glutamine; D006639:Histidine; D005972:Glutaminase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)