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The de novo synthesis of ornithine from glutamate by the rat small intestine has been investigated in order to establish conditions necessary for the reaction to proceed optimally. The formation of [ 14 C]ornithine from [ 14 C]glutamate was found in a subcellular fraction enriched in mitochondria. Ornithine formation required glutamate, ATP, NADPH, and Mg 2+ ; phosphocreatine in the presence of endogenous creatine phosphokinase was stimulatory. Glutamate and ATP exhibited typical saturation kinetics with apparent K m values of about 8 m m and 60 μ m , respectively. Ornithine synthesis was increased by NADPH concentrations up to 0.2 m m but higher concentrations were inhibitory. The maximal rate observed was 1 to 4 nmol of ornithine/min · mg protein. Synthesis of ornithine from glutamate was not observed with liver or kidney homogenates. Although the intestinal mitochondrial fraction contained ornithine aminotransferase (OAT), partially purified rat liver OAT stimulated ornithine synthesis. In the absence of exogenous liver OAT, pyridoxal 5′-phosphate or pyridoxamine 5′-phosphate stimulated both ornithine synthesis and endogenous OAT activity. These observations support the hypothesis that ATP and NADPH were utilized to reduce glutamate to glutamate-γ-semialdehyde which was then aminated by OAT to yield ornithine [Ross, G., Dunn, D., and Jones, M. E. (1978) Biochem. Biophys. Res. Commun. 85 , 140–147]. Pyridoxal 5′-phosphate concentrations greater than 0.01 m m inhibited ornithine formation whereas pyridoxamine 5′-phosphate showed no inhibitory effect at this or higher concentrations. Neither coenzyme inhibited endogenous OAT activity. These results suggest that pyridoxal 5′-phosphate inhibits the synthesis of glutamate-γ-semialdehyde.

A procedure for the purification of rat liver carbamyl phosphate synthetase (ammonia) to homogeneity is described. The molecular weight of isolated active enzyme is 222,000 ± 10,000; that of the subunits obtained by denaturation with sodium dodecyl sulfate in the presence of dithiothreitol is 120,000 ± 5,000. The enzyme appears to be composed of two subunits of identical size. The amino acid composition of the rat liver enzyme is reported and shown to be very similar to that of the bovine enzyme. The concentration of carbamyl phosphate synthetase I in the matrix of rat liver mitochondria is about 5 × 10 −4 m , very similar to that of acetylglutamate; on this basis, it is suggested that one-third to one-half of the total enzyme may be in the active form in the matrix. Calculations also indicate that normally, ATP is unlikely to limit the rate of carbamyl phosphate synthesis in vivo . Carbamyl phosphate synthetase I appears to constitute 22 to 26% of the total matrix protein of liver mitochondria; the significance of this fact is briefly examined.

Dihydroorotate dehydrogenase from rat liver is found to be located on the outer surface of the inner membrane of mitochondria. Dihydroorotate can diffuse freely from the cytosol into the mitochondria. Orotate can also diffuse freely from the mitochondria into the cytosol for futher conversion to UMP. Therefore, no active transport of either dihydroorotate or orotate is required in pyrimidine biosynthesis. The K m for l -dihydroorotate is 5.2 ± 0.6 μ m . pd -Dihydroorotate is not a substrate for the enzyme but is a competitive inhibitor with a K i of 1.4 m m . Of the compounds tested as analogs for dihydroorotate or metabolites related to pyrimidine biosynthesis, orotate is the strongest inhibitor, with a K i of 8.4 μ m . The K i values for 2,4-dinitrophenol and barbiturate are 180 and 56 μ m , respectively.

A study of microbial enzymes which catalyze carbamate formation was initiated. It is assumed that this enzyme activity is really a carbonic anhydrase since carbonic anhydrase catalyzes carbamate formation from ammonium bicarbonate solutions and since no other enzyme is known which does catalyze this reaction. We have studied principally the activity of yeast extracts. During attempts to purify the yeast enzyme it was found that this enzyme has associated with it two factors, small and dialyzable, which enhance the activity of crude extracts and ammonium sulfate fractions. One of the factors appears to be organic, and the other inorganic. Neither of these factors has any carbonic anhydrase activity by itself. Diamox (2-acetylamino-1,3,4-thiadiazole-5-sulfonamide), which inhibits bovine carbonic anhydrase 100% at 10−6 m , inhibits the yeast enzyme 50% at 10−2 m and 100% at 10−1 m .

Rabbit blood plasma was analyzed for the presence of carbamyl phosphate by incubating freshly collected plasma with aspartate-C 14 and aspartate transcarbamylase. No measurable concentration of carbamyl phosphate was found. Control experiments showed good recovery of added carbamyl phosphate so that it can be concluded that rabbit plasma contains less than 5 × 10 −6 M carbamyl phosphate. The implications of this finding with regard to the de novo synthesis of pyrimidines in mammals is discussed.

Crude extracts containing aspartate transcarbamylase (EC 2.1.3.2.) or purified preparations of aspartate transcarbamylase (ATCase) from ten bacteria have been analyzed by the gel filtration technique for molecular size (Andrews, 1964). Three size groups have been found which elute from Sephadex G-200 with Kd values of 0.13, 0.24, and 0.45. Pure Escherichia coli ATCase, whose molecular weight is 310,000 (Gerhart and Schachman, 1965), has a Kd of 0.25. Earlier studies on the feedback inhibition characteristics of bacterial ATCases by Neumann and Jones (1964) had predicted three types of bacterial ATCases. From the data available it is reasonable to assign each of the three size groups found here to one of the three kinetic groups found by Neumann and Jones (1964). The largest ATCases (Kd = 0.13, called A in this paper) are inhibited by pyrimidine nucleotides. The nucleotide inhibition is competitive toward the substrate carbamyl phosphate and noncompetitive toward aspartate. The substrate saturation curves for the Class A enzymes are hyperbolic in the absence of nucleotide. Upon the addition of a pyrimidine nucleotide, however, the carbamyl phosphate curve becomes sigmoidal. The small amount of kinetic data available on the ATCases which are of intermediate size (Kd = 0.24; called Class B in this paper) indicates that their activity is changed by pyrimidine nucleotide additions. However, with the exception of E. coli ATCase (Gerhart and Pardee, 1962 and 1964), their kinetic characteristics have not received much attention. The smallest ATCases (Kd = 0.45; called Class C in this paper) are not inhibited by pyrimidine nucleotides and have hyperbolic substrate curves. Crude extracts of two bacteria had both class B and a class C ATCases.The importance of these findings for bacterial taxonomy and for a more complete understanding of the regulation of pyrimidine biosynthesis as it occurs in bacteria, fungi, plants, and animals is briefly considered.

Factors influencing pyrroline 5-carboxylate (P5C) synthesis from glutamate by a subcellular fraction enriched in mitochondria of rat small intestinal mucosa have been studied. P5C synthesis decreased rapidly if this subcellular fraction was preincubated at 20 degrees C in the absence of substrates; this effect suggests that the enzyme(s) catalyzing P5C synthesis from glutamate (P5C synthase) is unstable in the absence of substrates. In the presence of substrates P5C synthesis increased linearly for the first 30 min of incubation, suggesting that the substrates promote enzyme stability. Pyridoxal 5'-phosphate is an effective inhibitor of P5C synthase whereas pyridoxamine 5'-phosphate and pyridoxal are not inhibitory. Potassium phosphate, KCl, and KBr each inhibited P5C synthase but potassium-Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) did not. Potassium phosphate was the most potent inhibitor followed by KBr, and then KCl. These results suggest P5C synthase is sensitive to anion inhibition. Both L-ornithine and D-ornithine inhibited P5C synthase; L-proline did not inhibit. Several analogs of ornithine and proline were also tested and none was found to inhibit P5C synthase; the inhibition by ornithine is, therefore, rather specific and it may prove to contribute to the regulation of metabolism of these amino acids.

The distribution of carbamyl phosphate synthetase (CPS) and/or ornithine transcarbamylase (OTC) was determined in 17 different rat tissues. Both enzymes, but particularly OTC, were found to be located mainly in the liver so that assay for OTC activity can provide a highly specific test for liver parenchymal cells. Optimal conditions for treatment of small samples of liver to maintain enzyme activity on storage are described. The significance of the absence of detectable CPS activity in most tissues for the presumed role of carbamyl phosphate in pyrimidine biosynthesis is discussed. In addition, it was found that (a) both CPS and OTC first appeared late in fetal life in the rat and did not reach adult levels until after birth, (b) OTC activity in rat and mouse hepatomas varied from undetectable to the normal level of adult liver, and (c) cells derived from liver and grown in culture were devoid of OTC activity.

N -carboxymethyl- N -acetyl- l -glutamic acid dimethylester (CM-AGAdiME) has been synthesized from acetyl- l -glutamic acid dimethylester and methyl chlorofor-mate. The synthesis of this compound was undertaken to allow us to ascertain if N -carboxy- N -acetyl- l -glutamate was formed as an intermediate in the reaction catalyzed by the ureotelic liver carbamyl phosphate synthetase. The latter substance was not detected in the enzyme reaction. When the diacylamine, CM-AGAdiME, is subject to hydroxylaminolysis at pH 6 or 14, acethydroxamate is formed as rapidly as with other acyl anhydrides; a result in keeping with the anhydride character of diacylamines. In an attempt to isolate N -acetyl- N -carboxyglutamic acid from CM-AGAdiME, the ester was subjected to liver aliesterase which removed quantitatively only one methyl group from the γ-carboxyl of CM-AGAdiME. The rapid decomposition of carboxymethylhydroxamate in acidic ferric chloride solutions is also described.

The activity of aspartate transcarbamylase (ATCase) from Escherichia coli is known to be regulated by feed-back inhibition. The catalytic and end-product inhibition characteristics of this enzyme from various species were compared. The enzyme from lettuce seedlings is best inhibited by uridylic acid. The inhibition is noncompetitive with regard to both aspartate and carbamyl phosphate, the substrates for the enzyme; it can be relieved specifically by ribose-5-phosphate without enzyme activity being impaired. The ATCase from Pseudomonas fluorescens is inhibited by various pyrimidine and purine derivatives, both in crude and 50-fold purified extracts. The most potent inhibitor is uridine-5′-triphosphate (UTP) but adenosine-5′-triphosphate also inhibits strongly. The degree of inhibition by UTP increases with decreasing carbamyl phosphate concentration and is independent of aspartate concentration. The enzyme could not be “desensitized” toward the inhibition by UTP by a variety of treatments without a parallel decrease in enzyme activity. ATCase from Bacillus subtilis is not inhibited by a large number of pyrimidine derivatives. The enzyme is inhibited by phosphate (product inhibition) competitively with regard to CAP. In this bacterium the synthesis of the ATCase is regulated by feed-back repression. The possible meaning of the differences in the characteristics of ATCase from the various organisms was discussed.

Studies were undertaken to investigate whether the natural co-factor of carbamyl-P synthetase, acetylglutamate, could act as a carrier for carbonate and carboxy-phosphate during carbamyl-P synthesis, as suggested earlier by Jones and Spector (1). For acetylglutamate to so act, either the α-amide N must lose a hydrogen or the carbonyl oxygen must enolize, which would result in a loss of a hydrogen from either the nitrogen or the acyl-methyl group. One approach to this problem is synthesis of appropriate analogues of acetylglutamate followed by a test of their ability to act as co-factor. Schooler et al. (2) have reported that 2-acetoxyglutarate is an effective co-factor for the enzyme, indicating that two of the above alternatives were unlikely. Their work is not, as yet, definitive and therefore requires confirmation, for they have not reported the method of synthesis used to prepare 2-acetoxyglutarate nor its complete characterization. This paper reports these details and confirms their observation that 2-acetoxyglutarate is an effective cofactor. In addition the ability of mono- and trifluoroacetyl glutamate to serve as co-factor is reported. A fourth analogue, 3-acetoxyglutarate will not serve as a cofactor. The third alternative i.e., enolization of the carbonyl group by the loss of a hydrogen atom of the acetyl-methyl group, was tested by experiments where the solvent was TOH. These experiments indicate that no hydrogen is labilized from the acetyl-methyl group. These results and earlier studies from a number of laboratories have completely tested the functional groups of active co-factors and consistently show that an active co-factor does not react with the other substrates (ammonia, bicarbonate, and ATP) required for carbamyl-P synthesis by carbamyl-P synthetase.