Search

Metabolic observations during early stages of hyperammonemia in two infants with ornithine transcarbamylase deficiency suggest that plasma alpha-ketoglutarate concentration ([alpha-KG]) becomes subnormal before the development of hyperammonemic coma. In one case, plasma [NH4+] remained normal until 40 days of age when it rose to 58 microM. However, this hyperammonemia was preceded by a fall in plasma [alpha-KG] to 15 microM at 27 days of age. It was only after severe hyperammonemia was established at 50 days of age that coma supervened. In the second case, plasma [alpha-KG] became subnormal (14 microM) 8 days before the rise in plasma ammonium concentration [NH4+] (52 microM) and 14 days before the onset of hyperammonemic coma. In eight patients with urea cycle enzymopathies, there was a highly significant (P less than 0.01) negative linear correlation between [NH4+] and [alpha-KG]. In patients with portal-systemic encephalopathy, there was a similar relationship between [NH4+] and [alpha-KG], although the absolute [alpha-KG] levels in these patients were normal (23 +/- 4 microM) while the patients were hyperammonemic (88 +/- 25 microM).

Ornithine carbamoyltransferase (EC 2.1.3.3) activity is a sensitive, specific indicator of hepatocellular injury. This paper describes development of an improved automated procedure for measurement of this activity. Triethanolamine-ethylenediaminetetraacetate is used as a buffer, and activity is determined by measuring the concentration of the product, citrulline. Kinetic studies have been performed to determine optimal pH and L-ornithine and carbamoyl phosphate concentrations. Recovery of citrulline was studied. The upper limit of normal obtained in a study of 106 blood-bank donors was 6 U/liter. The automated procedure developed as a result of these studies, in which optimal assay conditions are used, produces a threefold increase in sensitivity and permits use of a sample volume of 1 ml.

Eight enzymes involved in the conversion of acetylglutamate to arginine in Neurospora crassa were studied. The data indicate that of three enzymes early in the sequence, only the first, acetylglutamate kinase, is a nonorganellar enzyme. The next two, N-acetyl-gamma-glutamyl-phosphate reductase and acetylornithine aminotransferase, are in the mitochondrion, which was previously shown to contain the subsequent enzymes: acetylornithine-glutamate acetyltransferase, ornithine carbamyltransferase, and carbamyl-phosphate synthetase A (arginine specific). The last two enzymes of the pathway, argininosuccinate synthetase and argininosuccinate lyase, were previously shown to be cytosolic. All enzymes but one have low amplitudes or repression. Their levels respond little to arginine excess and are about twofold elevated (threefold for ornithine carbamyltransferase) as a result of arginine limitation in the arg-12-8 strain. No restriction of the incorporation of mitochondrial enzymes into mitochondria could be detected when the levels of these enzymes were elevated. Two enzymes, acetylglutamate kinase and carbamyl-phosphate synthetase A, which initiate the synthesis of the ornithine and guanidino moieties of arginine, respectively, show the lowest specific activities in crude extract. These enzymes display special regulatroy features. Acetylglutamate kinase, which has a typically low amplitude of repression, is subject to feedback inhibition. Carbamyl-phosphate synthetase A is wholly insensitive to arginine or citrulline in vitro or in vivo, but displays a very large amplitude of repression (about 60-fold). It is unique in that it can be almost completely repressed by growth of mycelia in excess arginine. These data suggest that mitochondrial localization may be incompatible with a mechanism of feedback inhibition by a cytosolic effector, arginine. Further, they suggest that the high repressibility of carbamyl-phosphate synthetase A compensates for its feedback insensitivity.

The presence of carbamyl-phosphate:glucose phosphotransferase in liver nuclei of five species of mammals and birds is demonstrated. The activity is confined to nuclear membranes and is due exclusively to multifunctional glucose-6-phosphatase-phosphotransferase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9). The nuclear enzyme constitutes approximately 16 to 19 percent of total hepatic glucose-6-phosphatase-phosphotransferase. Carbamyl-phosphate:glucose phosphotransferase and glucose-6-P phosphohydrolase activities of membrane of chicken liver nuclei are shown to be catalytically identical with the maximally activated microsomal enzyme. A correspondence is seen in two-substrate kinetic double reciprocal plots, K-m or apparent K-m values for the various substrates, K-i values for the competitive inhibitors P-i and ATP, and pH-activity profiles. Comparative studies were carried out with various intact, disrupted, and detergent-dispersed membranous preparations by a combination of enzyme kinetic and electron microscopic techniques. It is concluded that (a) intimate interrelationships exists between catalytic behavior of this enzyme and morphological integrity of membranes of which the enzyme is a part; (b) activities of the enzyme of nuclear membrane appear quite available for physiological phosphorylative functions; and (c) interrelationships between membrane morphology and catalytic behavior of this membrane-bound enzyme may well be involved in the bioregulation of this complex, multifunctional enzyme system.

Incubation of aqueous Ca2+, carbamylphosphate and ADP results in ATP formation with yields up to 20%. The dependence of the yield on the reaction parameters suggests that this transphosphorylation reaction proceeds heterogeneously on the surface of the calcium-carbamylphosphate precipitate.

Methylurea, a precursor of carcinogenic nitroso compound, was formed by incubating methylamine and carbamyl phosphate in neutral buffer. The formation was checked on a radiochromatogram, as well as by color reaction. The presence of methylamine and carbamyl phosphate in preserved, fermented foods provided a suitable condition for the formation of methylurea.

A radiochemical assay method of ornithine transcarbamylase (OTC) was developed using labeled carbamyl phosphate as a substrate. The enzyme activities determined by this method in peripheral white blood cells from ten normal subjects were 1.32 +/- 0.95 nmoles/mg/hr and the apparent Km's, when assayed at pH 8.5, were 6.4 mM for ornithine and 0.6 mM for carbamyl phosphate. On the contrary, the apparent Km's of human liver OTC were 0.6 mM for ornithine and 0.12 mM for carbamyl phosphate. The average OTC activity of granulocytes was 1.0 nmoles/mg/hr, whereas that of mononuclear cells was 0.4 mmoles/mg/hr. Lymphoid cell lines were established from three normal subjects and an OTC-deficient infant. All these cell lines demonstrated no OTC activity. When arginine was removed from the medium and replaced by ornithine, the lymphoid cells were unable to grow in culture. On autoradiography, the lymphoid cells showed labelling at incubation in the presence of 14C-citrulline, but not with 14C-ornithine.

The colorimetric method for phosphate determination described in the preceding paper is adapted for the assay of orthophosphate liberated in the aspartate transcarbamylase reaction. The method provides for simple, accurate, and sensitive measurement of enzyme activity. The assay uses ammonium molybdate and zinc acetate to form a colored complex with the enzymatically released phosphate; mild conditions which minimize the nonenzymatic background degradation of the substrate, carbamoyl phosphate, are used. Since the assay procedure is relatively rapid, it is especially attractive in situations where results are desired immediately. The method can be used for the assay of any enzyme which releases inorganic phosphate, even in the presence of labile organophosphate compounds.

Aspartate transcarbamylase from Escherichia coli has been prepared with up to four of zinc ions replaced by manganese, and the effect of this substitution on the proton nuclear magnetic resonance properties of succinate bound to the catalytic site and of cytidine 5'-triphosphate bound to the regulatory site has been determined, The specific activity and allosteric properties of the Mn-substituted enzyme are essentially identical with those of the native enzyme. The longitudinal relaxation time, T1, of the succinate protons is shortened by the native enzyme and is shortened further by the Mn-substituted enzyme at both 100 and 220 MHz in D2O solutions of 0.02 M immidazole chloride (pH 7.0), 10 minus 3 M beta-mercaptoethanol, 0;2 mM ethylenediamenetetraacetic acid, and 2.5 mM carbamyl phosphate over a temperature range of 5 to 35 degrees. Under the same conditions, the transverse relaxation time, T2, of the succinate protons at 90 MHz is shortened to the same extent by native and Mn-substituted enzyme. The temperature dependence of the relaxation times indicates that the shortening of the transverse relaxation time is determened by the lifetime of bound succinate, whereas the further shortening of the longitudinal relaxation time by the Mn-substituted enzyme is due to dipolar relaxation, i.e. to the interaction between Mn and the succinate protons. The distance between the Mn and the protons of succinate bound to the enzyme can be calculated from the relaxation time measurements and is 15,3 A. The dipolar interaction correlation time which is needed for the calculation of this distance, was found to be 3.5 X 10 minus 9 sec from the frequency dependence of T1. The transverse relaxation time of the C-6 proton of CTP is shortened to the same extent by both the native and Mn-substituted enzyme in D2O solutions of 0.02 M imidazole chloride (pH 7.0), 10 MINUS 3 M beta-mercaptoethanol, 0.2 mM ethylenediaminetetraacetic acid, and 2.5 mM carbamyl phosphate over the temperature 5-30 degrees. Since the temperature depencece of the relaxation time indicates the relaxation is not exchange limited, the manganese must be too distant from the bound CTP for an appreciable interaction to occur. This requires that the manganese be greater than 20A from the CTP. These results are used together with other available structural data to construct a schematic model for aspartate transcarbamylase.

With either alanine or a mixture of 15 different amino acids as nitrogen source, the addition of L-leucine inhibited the synthesis of urea by isolated rat liver cells. With alanine present leucine promoted the production of glutamate and glutamine. Comparison of effects of leucine on soluble glutamate dehydrogenase, mitochondria and isolated cells supports the postulate that leucine exerts its effect through activation of glutamate dehydrogenase. It is suggested that this latter enzyme may not be as important for the production of NH3 for carbamoyl phosphate synthesis as has been considered hitherto.

Mitochondria, microsomes, and cytosol all hydrolyze carbamylphosphate. Microsomes show the greatest specific activity. The mitochondrial enzyme is localized in the inner membranes, has optimum pH 5.5 and is heat-stable; the cytosol enzyme has optimum pH 5.0 and is heat-labile. Km for carbamylphosphate is 1.1 to 1.2 X 10(-2 M for both the cytosol and the mitochondrial enzyme. Both enzymes are inhibited by high substrate concentrations.

The result of binding the effectors ATP and CTP to aspartate transcarbamylase was studied by X-ray solution scattering. Binding of substrate analogues produces a substantial change in the solution scattering curve, allowing us to monitor the proportion of the different quaternary structure states present in solution. In the initial solution this ratio was made roughly unity by adding either carbamyl phosphate and succinate, or N-(phosphonacetyl)-L-aspartate (PALA). ATP or CTP were then added, and their effect on the proportion of the different quaternary structure states was followed. When using carbamyl phosphate and succinate (weakly bound), ATP or CTP had a clear effect, as observed previously by monitoring the sedimentation rate (Changeux et al., 1968). However, when PALA (strongly bound) was used, the effect of CTP was very much smaller, and that of ATP was undetectable. This result supports the explanation by Tauc et al. (1982), that nucleotides act mostly through changing the affinity of the active sites for substrate, and only to a small extent by directly modifying the quaternary structure equilibrium in the case of CTP.

The polar domains of the two transcarbamoylases, aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase, (OTCase) from Escherichia coli bind the common substrate carbamoyl phosphate and share extensive amino-acid sequence homology. The equatorial domains of the two enzymes differ in their substrate specificity (ATCase binds aspartate, OTCase binds ornithine) and have decreased sequence identity. While addressing the conservation of specific protein interactions during the evolution of these enzymes, we were able to switch one of their amino-acid-specific equatorial domains to produce a viable chimaeric enzyme. This was achieved by the in vitro fusion of DNA encoding the polar domain of OTCase to DNA encoding the equatorial domain of ATCase. The resulting gene fusion successfully transformed an argI-pyrB deletion strain of E. coli to pyrimidine prototrophy, giving rise to Pyr+ transformants that expressed ATCase but not OTCase activity. The formation of this active chimaeric enzyme shows that by exchanging protein domains between two functionally divergent enzymes we have achieved a switching in substrate specificity.

Several 13C isotope effects of relevance to reactions involving carbamate and carbamoyl phosphate have been determined. The fractionation factor of carbamate relative to aqueous CO2 is 1.011; the equilibrium isotope effect on the reaction catalyzed by carbamate kinase is 0.9983. From these data we can calculate that the fractionation factor of carbamoyl phosphate relative to aqueous CO2 is 1.013. The kinetic 13C isotope effect on the decomposition of carbamoyl phosphate to cyanate and phosphate is 1.058. The environment of the carbon atom in carbamate and carbamoyl phosphate and the mechanism of carbamoyl phosphate decomposition are discussed in light of these values.

Ornithine transcarbamylase, the enzyme which catalyzes the formation of citrulline from ornithine and carbamoylphosphate, has been purified from guinea pig liver. By the procedure indicated in the present paper a 200 fold purification of the enzyme has been achieved. Using both the purified fraction and the crude extract, a parallel determination of some physicochemical properties has been carried out. The pH of maximal activity of OTC was 7.8 for both preparations. The maximal stability of the enzyme with respect of pH showed a plateau over the range of pH 7 to 9.5 in the purified fraction, whereas the crude extract exhibited a major stability which lay between pH and 10. Both OTC preparations showed similar behavior regarding thermal stability, the enzyme being still active at a 50 degrees C temperature. The values of the apparent Km's proved to be 4.4 mM for the substrate ornithine and 5 mM for carbamoylphosphate.

Pyridoxal phosphate, a competitive inhibitor of aspartate transcarbamylase, binds to six sites in the catalytic and to twelve sites in the regulatory subunits of this hexameric protein. The properties of its association to the active sites of the enzyme are very similar to those observed with one of its substrates, carbamyl phosphate. It tightly binds to one half of the sites in the absence of succinate, an analogue of the second substrate. Since pyridoxal phosphate can be linked covalently to the protein by reduction, the distribution of the high affinity binding sites on catalytic trimers was studied after dissociation of modified holoenzyme. Electrophoresis of isolated subunits under non-denaturing conditions revealed four distinct bands, corresponding to trimers containing 0 to 3 pyridoxal phosphate derivatives. The distribution among the four species as a function of ligand concentration in the absence of succinate indicates that in the native oligomer, pyridoxal phosphate (and by extrapolation, carbamyl phosphate) binds to both catalytic trimers, rather than to three sites on a single subunit.

Mallavia, L. (University of Kansas, Lawrence) and D. Paretsky. Studies on the physiology of rickettsiae. V. Metabolism of carbamyl phosphate by Coxiella burnetii. J. Bacteriol. 86:232-238. 1963.-Preparations of disrupted Coxiella burnetii catalyze synthesis of citrulline from ornithine and carbamyl phosphate at an optimal pH of 7.0 to 7.5. Rickettsial synthesis of the pyrimidine precursor, ureidosuccinate, is demonstrated and confirmed by isolating C(14)-labeled ureidosuccinate from reaction mixtures of carbamyl phosphate and labeled aspartate. The data suggest a further rickettsial synthesis of orotate and imply rickettsial competence for host-independent pyrimidine synthesis.