Catalytic domains of carbamyl phosphate synthetase. Glutamine-hydrolyzing site of Escherichia coli carbamyl phosphate synthetase

We present evidence that cysteine 269 of the small subunit of Escherichia coli carbamyl phosphate synthetase is essential for the hydrolysis of glutamine. When cysteine 269 is replaced with glycine or with serine by site-directed mutagenesis of the carA gene, the resulting enzymes are unable to catalyze carbamyl phosphate synthesis with glutamine as nitrogen donor. Even though the glycine 269, and particularly the serine 269 enzyme bind significant amounts of glutamine, neither glycine 269 nor serine 269 can hydrolyze glutamine. The mutations at cysteine 269 do not affect carbamyl phosphate synthesis with NH3 as substrate. The NH3-dependent activity of the mutant enzymes was equal to that of wild-type. Measurements of Km indicate that the enzyme uses unionized NH3 rather than ammonium ion as substrate. The apparent Km for NH3 of the wild-type enzyme is calculated to be about 5 mM, independent of pH. The substitution of cysteine 269 with glycine or with serine results in a decrease of the apparent Km value for NH3 from 5 mM with the wild-type to 3.9 mM with the glycine, and 2.9 mM with the serine enzyme. Neither the glycine nor the serine mutation at position 269 affects the ability of the enzyme to catalyze ATP synthesis from ADP and carbamyl phosphate. Allosteric properties of the large subunit are also unaffected. However, substitution of cysteine 269 with glycine or with serine causes an 8- and 18-fold stimulation of HCO-3 -dependent ATPase activity, respectively. The increase in ATPase activity and the decrease in apparent Km for NH3 provide additional evidence for an interaction of the glutamine binding domain of the small subunit with one of the two known ATP sites of the large subunit.     

Alterations in the energetics of the carbamoyl phosphate synthetase reaction by site-directed modification of the essential sulfhydryl group

The change in reaction energetics of the bicarbonate-dependent ATPase reaction of Escherichia coli carbamoyl phosphate synthetase has been investigated for two site-directed mutations of the essential cysteine in the small subunit. Cysteine 269 has been proposed to facilitate the hydrolysis of glutamine by the formation of a glutamyl-thioester intermediate. The two mutant enzymes, C269S and C269G, along with the isolated large subunit, exhibit a 2-2.6-fold increase in the bicarbonate-dependent ATPase reaction relative to that observed for the wild type enzyme. In the presence of glutamine the overall enhancement is 3.7 and 9.0 for the C269G and C269S mutant enzymes, respectively. Carboxyphosphate is an intermediate in the bicarbonate-dependent ATPase reaction. The cause of the rate enhancements was investigated by measuring the positional isotope exchange rate in [gamma-18O4] ATP relative to the net rate of ATP hydrolysis. This ratio (Vex/Vchem) is a measure of the partitioning of the enzyme-carboxyphosphate-ADP complex. The partitioning ratio for the mutants is identical within experimental error to that observed for the wild type enzyme. This observation is consistent with the conclusion that the ground state for the enzyme-carboxyphosphate-ADP complex in the mutants is destabilized relative to the same complex in the wild type enzyme. If the increase in the absolute rate of ATP hydrolysis was due to a stabilization of the transition state for carboxyphosphate hydrolysis then the positional isotope exchange rate relative to the chemical hydrolysis rate would have been expected to decrease in the mutants.     

Identification of the junction between the glutamine amidotransferase and carbamyl phosphate synthetase domains of the mammalian CAD protein

The hamster CAD gene encodes a protein that catalyzes the first three steps of pyrimidine biosynthesis. We have sequenced a portion of a CAD cDNA and determined the location of the carbamyl phosphate synthetase II coding region. Subdomains coding for the glutamine hydrolyzing and carbamyl phosphate synthesizing functions have been identified through their high degree of similarity to carbamyl phosphate synthetase genes from a variety of organisms. The proline-rich junction between the glutaminase and synthetase domains, however, does not appear to be conserved among carbamyl phosphate synthetases.     

Identification of the reactive cysteinyl residue and ATP binding site in Bacillus cereus glutamine synthetase by chemical modification

Bacillus cereus glutamine synthetase was modified by reaction with a fluorescent SH reagent, N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS), or an ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The locations of the specific binding sites of these reagents were identified. IAEDANS inactivated Mg2(+)-dependent activity and activated Mn2(+)-dependent activity. FSBA inactivated only Mn2(+)-dependent activity. Mg2+ plus Mn2(+)-dependent activity was inactivated by IAEDANS or FSBA. Amino acid sequence analysis of the single AEDANS-labeled proteolytic fragment showed the cysteinyl residue at position 306 to be the site of modification. Cys 306 is one of three cysteines that are unique to Bacillus glutamine synthetase. The result suggested that the cysteine has a role in the active site of the enzyme. We also report that the amino acid residue modified by FSBA was the lysyl residue at position 43.     

Regulation of Escherichia coli carbamyl phosphate synthetase. Evidence for overlap of the allosteric nucleotide binding sites

Regulation of Escherichia coli carbamyl phosphate synthetase by UMP and IMP was examined in studies with various analogs of these nucleotides. Whereas UMP inhibits enzyme activity, the arabinose analog of UMP was found to be an activator. dUMP neither activates nor inhibits, but binds to the enzyme in a manner similar to UMP as evaluated by direct binding studies, sedimentation behavior, and ultraviolet difference spectral measurements. dUMP decreases inhibition by UMP and activation by IMP, but has no effect on activation by L-ornithine. The findings are in accord with the view that IMP and UMP bind to the same region of the enzyme; a possible general model for such overlapping binding sites is considered. Additional evidence is presented that inorganic phosphate can modulate regulation of the activity by nucleotides. Phosphate (and arsenate) markedly increase inhibition by UMP, decrease activation by IMP, but do not affect activation by L-ornithine. The extent of activation by IMP and by L-ornithine and that of inhibition by UMP are decreased when Mg2+ concentrations are increased relative to a fixed concentration of ATP. The findings suggest that the allosteric effectors may affect affinity of the enzyme for divalent metal ions as well as, as previously shown, the affinity of the enzyme for Mg-ATP.     

Autosomal recessive inheritance of human mitochondrial carbamyl phosphate synthetase deficiency.

The mode of inheritance of hepatic mitochondrial carbamyl phosphate synthetase (CPS I) deficiency has not been established conclusively in the past. In this study, hepatic tissue obtained by percutaneous biopsy from all members of the immediate family of two girls affected with partial CPS I deficiency was assayed for CPS I, ornithine transcarbamylase (OTC), and arginase activities. Only values for CPS I activity differed significantly from those in controls. The two affected girls each had markedly reduced CPS I activities of about 6% of the control mean. Their brother had activity well within the normal range. Of greatest significance was the finding that both parents had activities below the 95% confidence limits in controls, and intermediate between the deficient values of the two girls and the control range. The father and mother had, respectively, 32% and 54% of mean control activity. These data indicate that CPS I deficiency is inherited as an autosomal recessive trait and that the two affected girls are homozygous for the mutant gene, their brother is homozygous for the normal allele, and the parents are heterozygous.