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.     

Independent localization and regulation of carbamyl phosphate synthetase A polypeptides of Neurospora crassa

Summary Carbamyl phosphate synthetase A is a two-polypeptide, mitochondrial enzyme of arginine synthesis in Neurospora. The large subunit is encoded in the arg-3 locus and can catalyze formation of carbamyl-P with ammonia as the N donor. The small subunit is encoded in the unlinked arg-2 locus and imparts to the holoenzyme the ability to use glutamine, the biological substrate, as the N donor. By using nonsense mutations of arg-3, it was shown that the small subunit of the enzyme enters the mitochrondrion independently and is regulated in the same manner as it is in wild type. Similarly, arg-2 mutations, affecting the small subunit, have no effect on the localization or the regulation of the large subunit. The two subunits are regulated differently. Like most polypeptides of the pathway, the large subunit is not repressible and derepresses 3- to 5-fold upon argininestarvation of mycelia. In contrast, the glutamine-dependent activity of the holoenzyme is fully repressible and has a range of variation of over 100-fold. In keeping with this behavior, it is shown here that the small polypeptide, as visualized on two-dimensional gels, is also fully repressible. We conclude that the two subunits of the enzyme are localized independently, controlled independently and over different ranges, and that aggregation kinetics cannot alone explain the unusual regulatory amplitude of the native, two-subunit enzyme. The small subunit molecular weight was shown to be approximately 45,000.     

Evidence that mammalian glutamine-dependent carbamyl phosphate synthetase arose through gene fusion

Summary On the basis of homology, the mammalian CAD (glutamine-dependent carbamyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase) gene appears to have arisen from the fusion of four separate ancestral genes. Evidence for two of these precursor genes is found in the carbamyl phosphate synthetase (CPSase) domain of CAD. In prokaryotes, such as Escherichia coli CPSase is encoded by two distinct cistrons of the carAB operon. Whereas carA and carB are separated by a short noncoding intercistronic region, the homologous sequences of the CAD gene encode an amino acid bridge. This bridge connects the subdomains of the CAD CPSase. We constructed a bacterial carAB fusion gene in which the intercistronic region codes for a hamster bridgelike sequence. The fused carAB gene directs the synthesis of a stable bifunctional polypeptide whose glutamine-dependent CPSase activity is comparable to the E. coli CPSase holoenzyme. The fusion in E. coli of the single gene counterparts of CAD demonstrates a potential model system to study the genetic events that lead to gene fusion and the creation of multienzymatic proteins. Offprint requests to: J.N. Davidson     

A rapid colorimetric assay for carbamyl phosphate synthetase I

A rapid, reproducible, and sensitive colorimetric assay for carbamyl phosphate synthetase I was presented. A four-fold increase in sensitivity and reduced assay time were afforded by this procedure. The method utilized the chemical conversion of carbamyl phosphate to hydroxyurea by the action of hydroxylamine instead of employing a coupling enzyme. The hydroxyurea was quantitated in 15 min by an improved colorimetric assay for ureido compounds by measuring the absorption of the resulting chromophore at 458 nm. Optimum conditions for both the formation and quantitation of hydroxyurea were established. Activity measurements of carbamyl phosphate synthetase I obtained by this uncoupled method were identical with those obtained by the ornithine transcarbamylase coupld assay.     

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.     

Genetic analysis of carbamyl phosphate synthetase I deficiency

Summary Carbamyl phosphate synthetase I deficiency (CPSD) is an autosomal recessive disorder of ureagenesis characterized by hyperammonemic coma in the neonatal period. To study the genetic basis of CPSD we have performed a molecular analysis of the CPS I genes in CPSD patients from six unrelated families. Using a cDNA probe for the human CPS I gene and restriction endonuclease mapping techniques, we observed no abnormality in the number or size of the hybridizing DNA fragments from the seven affected individuals examined. These findings suggest that no gross alteration affected the CPS I genes. We did detect a frequent restriction fragment length polymorphism (RFLP) at the CPS I locus which we employed as a linkage marker. Our results suggest the polymorphic CPS I restriction fragments cosegregate with the CPSD phenotype, and that linkage disequilibrium exists between the CPSI RFLPs studied and the affected alleles. The RFLPs described may enable prenatal detection of CPSD in families where the coupling phases between CPSD alleles and RFLPs can be determined.A preliminary report of these studies was presented at the Society for Pediatric Research meetings, San Francisco, May 1984 and appeared in abstract form in Pediatric Research 18:296A (1984)     

In vivo synthesis of carbamyl phosphate from NH3 by the large subunit of Escherichia coli carbamyl phosphate synthetase

The cloned carAB operon of Escherichia coli coding for the small and large subunits of carbamyl phosphate synthetase has been used to construct a recombinant plasmid with a 4.16 kilobase ClaI fragment of the car operon that lacks the major promoters, P1 and P2. The plasmid, pHN12, carries a functional carB gene. A mutant E. coli strain lacking both subunits of carbamyl phosphate synthetase when transformed with pHN12 overproduces the large subunit by 200-fold (8-10% of the cellular protein). The elevated levels of the large subunit enable the transformed cells to utilize NH3 but not glutamine as nitrogen donor for carbamyl phosphate synthesis. The large subunit has been purified from the overexpressing strain. The purified native large subunit is capable of synthesizing carbamyl phosphate from ammonia, HCO-3, and ATP. The kinetic properties of the large subunit compared with the holoenzyme indicate that the Michaelis constants of the large subunit for HCO-3 and ATP are modulated by its association with the small glutamine binding subunit.     

A major polypeptide component of rat liver mitochondria: carbamyl phosphate synthetase.

One of the major components of rat liver mitochondria detected by gel electrophoresis in sodium dodecyl sulfate is a 165,000 molecular weight polypeptide that makes up 15 to 20% of the total mitochondrial protein. This component appears to be a single molecular species. Evidence is presented here for the identification of this protein with the polypeptide chain of a urea cycle enzyme, carbamoylphosphate synthetase I (EC 2.7.2.5). The 165,000 molecular weight polypeptide was solubilized from mitochondria with Triton X-100 and purified to 90% homogeneity by DEAE-cellulose chromatography. This component co-migrated with carbamyl phosphate synthetase activity when mitochondrial proteins were separated by gel filtration or sucrose gradient centifugation. The identification of the 165,000 molecular weight polypeptide with this activity was also supported by the presence or absence of this protein in a variety of rat tissue mitochondria, in liver and kidney mitochondria from various ureotelic and nonureotelic species, and in fetal rat liver mitochondria.