Carbamyl phosphate biosynthesis in Bacillus subtilis

Issaly, Inda M.; Issaly, A. S.; Reissig, José L.

doi:10.1016/0005-2744(70)90126-9

Cited by 24 publications

(19 citation statements)

References 24 publications

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“…It is interesting to note that in Bacillus subtili8, a single mutational event leads to a decrease of both the glutaminedependent carbamyl phosphate synthetase and carbamate kinase activities (16). This result may be integrated with the present findings if it is assumed that the carbamate kinase activity is catalyzed by the heavy chain of carbamyl phosphate synthetase, and if the heavy chain is synthesized in this organism in excess of the light chain.…”

Section: Discussionsupporting

confidence: 82%

Reversible Dissociation of Carbamyl Phosphate Synthetase into a Regulated Synthesis Subunit and a Subunit Required for Glutamine Utilization

Trotta

Burt

Haschemeyer

et al. 1971

Proc. Natl. Acad. Sci. U.S.A.

153

118

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Carbamyl phosphate synthetase (from Escherichia coli) consists of a 7.3S protomeric unit that contains one heavy polypeptide chain (molecular weight about 130,000) and one light chain (molecular weight about 42,000). The heavy and light chains were separated by gel filtration in the presence of 1 M potassium thiocyanate. In contrast to the native enzyme and the reconstituted enzyme (prepared by mixing the separated heavy and light chains), the heavy chain does not catalyze glutamine-dependent carbamyl phosphate synthesis, although it does catalyze the synthesis of carbamyl phosphate from ammonia. The heavy chain also catalyzes two of the partial reactions catalyzed by the intact enzyme; i.e., the bicarbonate-dependent cleavage of ATP and the synthesis of ATP from ADP and carbamyl phosphate. Both positive (ammonia, ornithine, IMP) and negative (UMP) allosteric regulatory sites are located on the heavy chain. The only catalytic activity exhibited by the light chain is the hydrolysis of glutamine. A model is presented according to which glutamine binds to the light chain, which is followed by release of nitrogen from the amide group for use by the heavy chain. The findings suggest that glutamine-dependent carbamyl phosphate synthetase (and perhaps other glutamine amidotransferases) arose in the course of evolution by a combination of a primitive ammonia-dependent synthetic enzyme and a glutaminase; this combination may have been associated with a change from ammonia to glutamine as the principal source of nitrogen.Carbamyl phosphate, a metabolite of broad biological significance, is required for the biosynthesis of the pyrimidines and of the amino acid arginine. There are two types of carbamyl phosphate synthetases (1-3). One of these uses ammonia and requires N-acetylglutamate as a cofactor. The other can use both glutamine and ammonia and does not require N-acetylglutamate; glutamine is thought to be the "natural" substrate because it exhibits a higher affinity for the enzyme. Enzymes of the latter type resemble the other glutamine amidotransferases, which catalyze reactions in which the amide group of glutamine is used for various synthetic purposes; these enzymes can also use ammonia in place of glutamine (4).The glutamine-dependent carbamyl phosphate synthetase of Escherichia coli is subject to positive and negative feedback control by products of the purine and pyrimidine biosynthetic pathways, respectively (5), as well as by positive regulation by ornithine (6) and ammonia (unpublished It has recently been demonstrated that the enzyme can be dissociated into two kinds of polypeptide chains under extreme denaturing conditions*. We have independently made similar observationst, and now report that a relatively mild solvent perturbation promotes a reversible dissociation into nonidentical subunits. We were thus able to physically separate the two subunits and to compare their regulatory and catalytic properties with those of the recombined system and of the original enzyme.

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Section: Discussionsupporting

confidence: 82%

Reversible Dissociation of Carbamyl Phosphate Synthetase into a Regulated Synthesis Subunit and a Subunit Required for Glutamine Utilization

Trotta

Burt

Haschemeyer

et al. 1971

Proc. Natl. Acad. Sci. U.S.A.

153

118

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“…Under the conditions of arginine starvation and low concentration of glucose described above, the synthesis of the ornithine carbamoyltransferase was derepressed 4-5-fold and it was not subject to catabolite repression [12].…”

Section: Partial Purification O J Ornithine Carbamoyltransferasementioning

confidence: 99%

“…The experiments were performed with an arginine auxotrophic mutant (arg), obtained by us [7] from a strain of B. subtilis try C2, kindly provided by Dr P. Schaeffer.…”

Section: Strain Media and Culturesmentioning

confidence: 99%

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Control of Ornithine Carbamoyltransferase Activity by Arginase in Bacillus subtilis

Issaly¹,

Issaly²

1974

European Journal of Biochemistry

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Partially purified Bacillus subtilis ornithine carbamoyltransferase is cooperatively inhibited by excess of ornithine. The inhibition is decreased by lowering of pH from 9.2 to 7.6 without altering the affinity of the enzyme for the neutral species of ornithine which seems to be its true substrate.Arginine and lysine partially reverse this inhibition without affecting the catalytic activity of the enzyme. 2-Aminobutyrate, an inhibitor of the enzyme, competitive with respect to ornithine, is not able to replace ornithine in this excess-substrate type of inhibition. Zn2+ is also an inhibitor of ornithine carbamoyltransferase, with competitive action towards ornithine and its inhibition is added to the excess ornithine inhibition. These data suggest that ornithine carbamoyltransferase has a regulatory site for ornithine, distinct from the catalytic one.Partially purified arginase, prepared from the same strain, increases the inhibition of ornithine carbamoyltransferase by excess of ornithine when arginine is present. An association of the two enzymes is suggested by molecular sieving. These data postulate that ornithine carbamoyltransferase is regulated by arginase under the control of arginine and ornithine. The other substrate, carbamoyl phosphate, is not involved in this regulatory mechanism.Lysine and Zn2 + inhibit arginase competitively with respect to arginine without altering the effect of this enzyme on the inhibition of ornithine carbamoyltransferase. 2-Aminobutyrate inhibits arginase non-competitively with respect to arginine and prevents the inhibition of arginase towards ornithine carbamoyltransferase. These results suggest that arginase possesses a regulatory site for arginine, distinct from the catalytic one, which is also involved in the control mechanism.Wiame et al. [l-31 have uncovered a novel mechanism of metabolic regulation in the arginine cycle of yeasts. This mechanism, for which the name "epienzymatic control" has been coined [2], results from the binding of ornithine carbamoyltransferase with arginase, under the control of arginine and ornithine, forming a complex in which the ornithine carbamoyltransferase is strongly inhibited without substantial modification of the arginase activity. This control mechanism ensures that the arginine cycle will not spin aimlessly, making and destroying arginine with a net loss of energy.Aas a step towards exploring the generality of epienzymatic control mechanism, it appeared of interest to establish whether they also occur in prokaryotes. The present study will show that, indeed, the ornithine carbamoyltransferase of Bacillus subtilis is Enzymes.

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“…The glutamine-dependent enzyme apparently possesses an anabolic role (5,15). The presence of both carbamate kinase and CAP synthetase in Bacillus subtilis has been recently reported (7). Fungi appear to have two CAP synthetases; one, specific for pyrimidine synthesis, is controlled through repression and end product inhibition by the pyrimidine pool (11,12,19), and one, specific for arginine, is repressed and inhibited by arginine (5,11).…”

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confidence: 99%

Effect of Urea on Ammonium-dependent Synthesis of Carbamyl Phosphate during Spore Germination of Geotrichum candidum

1972

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The specific activities of enzymes catalyzing the ammoniumdependent carbamyl phosphate synthesis (NHa-CPS) and the glutamine-dependent carbamyl phosphate synthesis (GLN-CPS) were increased during germination by approximately 5-and 1.7-fold respectively in the presence of 35 mM urea. The increase of NH3-CPS and GLN-CPS levels occurred immediately after the onset of germination and prior to the appearance of germ tube. Ammonium also stimulated the NH3-CPS activity, but the induction caused by urea was about three times higher than that by ammonium. (5,15). The presence of both carbamate kinase and CAP synthetase in Bacillus subtilis has been recently reported (7). Fungi appear to have two CAP synthetases; one, specific for pyrimidine synthesis, is controlled through repression and end product inhibition by the pyrimidine pool (11,12,19), and one, specific for arginine, is repressed and inhibited by arginine (5,11). Both NH3-CPS and GLN-CPSSpores of Geotrichuin candidum lack urease, yet urea is readily metabolized during germination by means of ATP and urea amidolyase (18). Previous studies have shown that addition of urea to the germinating medium of G. candidum results 'Abbreviations: CAP: carbamyl phosphate; DEAE: diethylaminoethyl.in derepression of ornithine transcarbamylase (2). The present work describes the effect of urea on the NHr-dependent synthesis of CAP in relation to the possible physiological function of the latter reaction in G. candidum. MATERIALS AND METHODSProcedures for obtaining and germinating conidia of G. candidum were described (1). The germination medium (YEG) contained 0.1% yeast extract, 0.5% glucose, and 50 mm phosphate buffer, pH 7.5. The germinated spores were harvested (18) and either used immediately for determination of endogenous ammonia or maintained frozen until used for preparation of cell-free extracts. Enzyme extraction was performed according to Barash and Mor (2) in 100 mm tris-HCl buffer, pH 7.8, which contained 20 mm MgClh, 10 mm 2-mercaptoethanol, and 10% v/v glycerol. Enzyme assays in the crude extracts were carried out immediately following dialysis of 3 hr against the extraction buffer.When enzyme fractionation was required, the crude extract was centrifuged at 105,000g for 45 min, and the enzyme was precipitated by ammonium sulfate between 25 and 50% saturation. The precipitate was dissolved in 5 to 10 ml of the extraction buffer and dialyzed against the same buffer for 3 hr. The enzyme preparation was then placed on a column (2 X 15 cm) of DEAE-cellulose (Whatman microgranular previously equilibrated with 50 mm tris-HCl, pH 8.0, containing 10 mM 2-mercaptoethanol, 20 mM MgCl2, and 10% v/v glycerol. Enzyme elution was carried out by linear increase in KCl concentration from 0 to 0.5 M.The enzymatic synthesis of CAP was determined by both colorimetric and radioactive methods. The assay system for NHv-dependent CAP synthesis (NH,-CPS), using the colorimetric method, contained (in a total volume of 1.5 ml): ATP, 10 umoles; MgCl2, 20 ,umoles; tris-HC1 buffer, ...

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Carbamyl phosphate biosynthesis in Bacillus subtilis

Cited by 24 publications

References 24 publications

Reversible Dissociation of Carbamyl Phosphate Synthetase into a Regulated Synthesis Subunit and a Subunit Required for Glutamine Utilization

Reversible Dissociation of Carbamyl Phosphate Synthetase into a Regulated Synthesis Subunit and a Subunit Required for Glutamine Utilization

Control of Ornithine Carbamoyltransferase Activity by Arginase in Bacillus subtilis

Effect of Urea on Ammonium-dependent Synthesis of Carbamyl Phosphate during Spore Germination of Geotrichum candidum

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