Pyrroline-5-carboxylate synthesis from glutamate by rat intestinal mucosa. Subcellular localization and temperature stability

The demonstration of the ornithine biosynthesis from glutamate in cell-free homogenates of rat intestinal mucosa by Ross, G., Dunn, D., and Jones, M.E. (1978) Biochem. Biophys. Res. Commun. 85, 140-147 suggested that this tissue might have the capacity to convert glutamate to pyrroline-5-carboxylate (P5C). We have shown in the preceding paper (Wakabayashi, Y., and Jones, M.E. (1983) J. Biol. Chem. 258, 3865-3872) that this is the case. The intracellular distribution of the P5C-synthesizing activity was investigated utilizing a newly developed procedure for subcellular fractionation of the rat intestinal mucosa. We found that the activity resided in the mitochondrial fraction as characterized by marker enzymes and an electron micrograph. The mitochondrial membrane fraction, freed of the soluble matrix and intermembrane space enzymes, retained all of the P5C-synthesizing activity. Addition of the soluble fraction to the membrane fraction did not affect the activity. P5C synthase, the name we have chosen for the protein(s) that catalyzes P5C synthesis from glutamate when ATP and NADPH are present, is susceptible to thermal inactivation in the presence of detergent. By lowering the incubation temperature to or below 20 degrees C, one can obtain a linear production of P5C with respect to time and protein concentration. Lower incubation temperatures are recommended for routine assay of this enzyme(s). Addition of 30% glycerol to the incubation mixture resulted in a linear formation of P5C with time at 30 degrees C; this and other data suggest that polyhydroxylic compounds may protect this protein against denaturation. Preliminary experiments suggest that P5C synthase can be extracted from a mitochondrial membrane in the presence of detergent, a high salt concentration, and glycerol. The possibility that the enzyme(s) is located in the inner mitochondrial membrane is discussed.     

The biosynthesis of sulphogalactosyldiacylglycerol of rat brain in vitro

Triton X-100 extracts of rat brain microsomal fraction catalyse the formation of sulphogalactosyldiacylglycerol from galactosyldiacylglycerol and adenosine 3'-phosphate 5'-sulphatophosphate. Of the various subcellular fractions of brain assayed, the microsomal fraction contained most (79%) of the adenosine 3'-phosphate 5'-sulphatophosphate-galactosyldiacylglycerol sulphotransferase activity. The enzyme activity was stimulated by Triton X-100 and showed linearity with increasing time, concentrations of enzyme and added substrates. ATP and KF prolonged the linearity of the activity with time, but ATP had an overall inhibitory effect on the sulphotransferase. Both ATP and KF inhibit the degradation of adenosine 3'-phosphate 5'-sulphatophosphate, which probably causes the increased linearity of the sulphotransferase reaction with time. The enzyme preparation did not catalyse the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to either cholesterol or galabiosyldiacylglycerol (galactosylgalactosyldiacylglycerol). Significant differences between the formation of sulphogalactosyldiacylglycerol and cerebroside sulphate catalysed by the same enzyme preparation were noted. ATP and Mg(2+) strongly inhibit the formation of sulphogalactosyldiacylglycerol but equally strongly stimulate the synthesis of cerebroside sulphate. The apparent K(m) for galactosyldiacylglycerol is 200mum, and that for cerebroside is 45mum. Galactosyldiacylglycerol and cerebroside are mutually inhibitory toward the synthesis of sulphated derivatives of each. These data do not necessarily lead to the conclusion that two sulphotransferases are present, but they do indicate a possible means of controlling the synthesis of these two sulpholipids.     

Nutritional and metabolic interrelationships of arginine, glutamic acid and proline in the chicken.

Proline satisfies by a narrow margin the criterion for dietary essentially for the chick. It is estimated that the chick may synthesize 80-90% of the total proline needed for growth. Although the metabolism of arginine, ornithine and glutamic acid is expected to give rise to proline, dietary supplements to these amino acids are relatively ineffective in reducing the proline requirement of chicks. Studies of the efficacy of dietary ornithine for growth, and tracer studies using L-(5-3H)arginine indicate that the conversion of ornithine to proline in vivo is limited, and the amount of proline synthesized from arginine is but a small fraction of that needed for growth. The limiting processes in proline synthesis from glutamic acid and ornithine are not known. In Escherichia coli, where the biosynthetic pathway from glutamate to proline has been elucidated, a glutamate kinase, NADP-dependent delta1-pyrroline-5-carboxylic acid (P5C) dehydrogenase and P5C reductase catalyze proline synthesis. P5C reductase is present in the soluble fraction of chicken liver and kidney. An NADP-dependent P5C dehydrogenase activity has also been observed in this fraction of liver. Further studies are required to assess the importance of these enzymes in proline biosynthesis and to determine the limiting process in proline formation in the chicken.     

Effects of inhibition of ornithine aminotransferase or of general aminotransferases on urea and citrulline synthesis and on the levels of acetylglutamate in isolated rat hepatocytes

Summary Canaline and gabaculine, inhibitors of γ-aminotransferases and thus of ornithine aminotransferase (E.C. 2.6.1.13), decreased the flow through ornithine carbamoyl transferase (E.C. 2.1.3.3) in isolated rat hepatocytes incubated with 10 mM NH₄Cl and ornithine. The levels of acetylglutamate, an essential activator of carbamoyl phosphate synthetase (ammonia) (E.C. 6.3.4.16), were also decreased, suggesting that the inhibitors had also caused a decrease in the rate of carbamoyl phosphate synthesis. Under these conditions, ornithine appears to be a precursor of acetylglutamate, via ornithine aminotransferase, possibly as a consequence of glutamate synthesis. The influence of aminooxyacetate, an aminotransferase inhibitor, has also been examined.     

Regulation of arginine and pyrimidine biosynthesis in Pseudomonas putida.

Repression of biosynthetic enzyme synthesis in Pseudomonas putida is incomplete even when the bacteria are growing in a nutritionally complex environment. The synthesis of four of the enzymes of the arginine biosynthetic pathway (N-acetyl-alpha-glutamokinase/N-acetylglutamate-gamma-semialdehyde dehydrogenase, ornithine carbamoyltransferase and acetylornithine-delta-transaminase) could be repressed and derepressed, but the maximum difference observed between repressed and derepressed levels for any enzyme of the pathway was only 5-fold (for ornithine carbamoyltransferase). No repression of five enzymes of the pyrimidine biosynthetic pathway (aspartate carbamoyltransferase, dihydro-orotase, dihydro-orotate dehydrogenase, orotidine-5'-phosphate pyrophosphorylase and orotidine-5'-phosphate decarboxylase) could be detected on addition of pyrimidines to minimal asparagine cultures of P. putida A90, but a 1-5- to 2-fold degree of derepression was found following pyrimidine starvation of pyrimidine auxotrophic mutants of P. putida A90. Aspartate carbamoyltransferase in crude extracts of P. putida A90 was inhibited in vitro by (in order of efficiency) pyrophosphate, CTP, UTP and ATP, at limiting but not at saturating concentrations of carbamoyl phosphate.     

Substrate utilization by Legionella cells after cryopreservation in phosphate buffer

The objective of this study was to evaluate by relatively simple metabolic tests the usefulness of buffers and energy sources commonly used in Legionella growth media. Legionella pneumophila serogroups 1 to 6, Legionella micdadei, and Legionella bozemanii were grown in an enriched charcoal-yeast extract diphasic medium. The cells were washed thrice, suspended in various buffers (pH 6.9) with 1 or 5 mM MgSO4, and used immediately or after controlled-rate cryopreservation. CO2 produced and C incorporated into the cold trichloracetic acid-insoluble fractions from 14C-labeled substrates were determine. Potassium phosphate buffer (0.02 M) was as satisfactory as organic buffers for glutamate metabolism, but the addition of KCl or NaCl reduced activity. Metabolic activity for glutamate was not lost upon cryopreservation, and cryopreserved cells were used to test the utilization of other single or paired substrates. Rates of activity for serine, glutamate, threonine, and pyruvate, in this descending order, were high, and those for alpha-ketoglutarate, succinate, and gamma-aminobutyrate were low. Although glutamine was not used as rapidly as glutamate, when added to glutamate it was preferentially metabolized, possibly because of more rapid transport. When glutamate and serine were combined, glutamate furnished more C for CO2 and less for incorporation, whereas the reverse was true of serine. In conclusion, glutamate as an energy source may in some cases spare other amino acids for synthesis. alpha-Ketoglutarate, a common constituent of Legionella media, may reduce oxygen toxicity but is probably not a chief energy source.     

Substrate utilization by Legionella cells after cryopreservation in phosphate buffer.

The objective of this study was to evaluate by relatively simple metabolic tests the usefulness of buffers and energy sources commonly used in Legionella growth media. Legionella pneumophila serogroups 1 to 6, Legionella micdadei, and Legionella bozemanii were grown in an enriched charcoal-yeast extract diphasic medium. The cells were washed thrice, suspended in various buffers (pH 6.9) with 1 or 5 mM MgSO4, and used immediately or after controlled-rate cryopreservation. CO2 produced and C incorporated into the cold trichloracetic acid-insoluble fractions from 14C-labeled substrates were determine. Potassium phosphate buffer (0.02 M) was as satisfactory as organic buffers for glutamate metabolism, but the addition of KCl or NaCl reduced activity. Metabolic activity for glutamate was not lost upon cryopreservation, and cryopreserved cells were used to test the utilization of other single or paired substrates. Rates of activity for serine, glutamate, threonine, and pyruvate, in this descending order, were high, and those for alpha-ketoglutarate, succinate, and gamma-aminobutyrate were low. Although glutamine was not used as rapidly as glutamate, when added to glutamate it was preferentially metabolized, possibly because of more rapid transport. When glutamate and serine were combined, glutamate furnished more C for CO2 and less for incorporation, whereas the reverse was true of serine. In conclusion, glutamate as an energy source may in some cases spare other amino acids for synthesis. alpha-Ketoglutarate, a common constituent of Legionella media, may reduce oxygen toxicity but is probably not a chief energy source.     

Solubilization of 1,25-dihydroxyvitamin D3 receptor with pyridoxal 5'-phosphate from hen intestinal mucosa

1,25-Dihydroxyvitamin D3(1,25-(OH)2D3) receptor was solubilized in cytosol fractions upon homogenization of hen intestinal mucosa with pyridoxal 5'-phosphate contained in a low ionic strength buffer. Pyridoxal 5'-phosphate did not inhibit the binding of 1,25-(OH)2D3 to its receptor. The receptor solubilized with pyridoxal 5'-phosphate was similar to the KCl-solubilized receptor in its binding affinity to the hormone and sedimentation coefficient. A majority (greater than 90%) of the mucosal 1,25-(OH)2D3 receptors were obtained as associating with crude chromatin which was prepared with a low ionic strength buffer, and this fraction of the receptor was solubilized with pyridoxal 5'-phosphate. Ten millimolar pyridoxal 5'-phosphate was as effective as approx 0.2 M KCl in solubilizing the receptor from the crude chromatin. Pyridoxal 5'-phosphate also showed a potency to dissociate the 1,25-(OH)2D3-receptor complex previously bound to DNA-cellulose. Pyridoxal 5'-phosphate-related compounds such as pyridoxamine 5'-phosphate and pyridoxal did not show this potency. These results suggest that pyridoxal 5'-phosphate reduced the interaction of 1,25-(OH)2D3 receptor with its nuclear binding components without inhibiting the binding of the receptor to the hormone.     

Carbon Dioxide Fixation by Extracts of Streptococcus faecalis var. liquefaciens

Extracts of cells of Streptococcus faecalis var. liquefaciens strain 31 incorporated (14)CO(2) into aspartate. Dialyzed extracts produced radioactive oxalacetate in the absence of exogenously added glutamate and pyridoxal-5'-phosphate and produced radioactive aspartate in the presence of these components. Reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate could not be substituted for adenosine triphosphate (ATP); phosphoenolpyruvate even in the presence of nucleoside diphosphates could not replace pyruvate plus ATP; propionate plus coenzyme A (CoA) could not replace pyruvate in supporting CO(2) fixation by cell extracts. Fixation by dialyzed cell extracts required pyruvate, ATP, MgSO(4), and was stimulated by biotin, KCl, 2-mercaptoethanol, CoA, and acetyl CoA. Inhibition of fixation occurred when avidin, NaCl, oxalacetate, or aspartate was added to dialyzed extracts. On the basis of the products formed and the effects of substrates and cofactors on the fixation reaction, it was concluded that pyruvate carboxylase is responsible for CO(2) fixation in this microorganism.     

Synthesis and evaluation of a mechanism-based inhibitor of KDO8P synthase

The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and D-arabinose 5-phosphate (A5P) to produce KDO8P and inorganic phosphate. In attempts to investigate the lack of antibacterial activity of the most potent inhibitor of KDO8P synthase, the amino phosphonophosphate 3, we have synthesized its hydrolytically stable isosteric phosphonate analogue 4 and tested it as an inhibitor of the enzyme. The synthesis of 4 was accomplished in a one step procedure by employing the direct reductive amination in aqueous media between unprotected sugar phosphonate and glyphosate. The analogue 4 proved to be a competitive inhibitor of KDO8P synthase with respect to both substrates A5P and PEP binding. In vitro antibacterial tests against a series of different Gram-negative organisms establish that both inhibitors (3 and 4) lack antibacterial activity probably due to their reduced ability to penetrate the bacterial cell membrane.     

Structural studies of the catalytic reaction pathway of a hyperthermophilic histidinol-phosphate aminotransferase

In histidine biosynthesis, histidinol-phosphate aminotransferase catalyzes the transfer of the amino group from glutamate to imidazole acetol-phosphate producing 2-oxoglutarate and histidinol phosphate. In some organisms such as the hyperthermophile Thermotoga maritima, specific tyrosine and aromatic amino acid transaminases have not been identified to date, suggesting an additional role for histidinol-phosphate aminotransferase in other transamination reactions generating aromatic amino acids. To gain insight into the specific function of this transaminase, we have determined its crystal structure in the absence of any ligand except phosphate, in the presence of covalently bound pyridoxal 5'-phosphate, of the coenzyme histidinol phosphate adduct, and of pyridoxamine 5'-phosphate. The enzyme accepts histidinol phosphate, tyrosine, tryptophan, and phenylalanine, but not histidine, as substrates. The structures provide a model of how these different substrates could be accommodated by histidinol-phosphate aminotransferase. Some of the structural features of the enzyme are more preserved between the T. maritima enzyme and a related threonine-phosphate decarboxylase from S. typhimurium than with histidinol-phosphate aminotransferases from different organisms.     

Altered enzyme activities and citrulline synthesis in liver mitochondria from ornithine carbamoyltransferase-deficient sparse-furash mice.

Male mice carrying the spfash mutation have 5-10% of the normal activity of ornithine carbamoyltransferase, yet are only slightly hyperammonaemic and develop quite well. A study of liver mitochondria from normal and spfash males showed that they differ in important ways. (1) The spfash liver contains about 33% more mitochondrial protein per g than does normal liver. (2) The specific activities of carbamoyl-phosphate synthetase (ammonia) and glutamate dehydrogenase are about 15% lower than normal in mitochondria from spfash mice, whereas those of beta-hydroxybutyrate dehydrogenase and cytochrome oxidase are 22% higher and 30% lower respectively. (3) In the presence of 10 mM-ornithine and the substrates for carbamoyl phosphate synthesis, coupled and uncoupled mitochondria from spfash mice synthesize citrulline at unexpectedly high rates, about 25 and 44 nmol/min per mg respectively. Though these are somewhat lower than the corresponding rates obtained with normal mitochondria, the difference does not arise from the deficiency in ornithine carbamoyltransferase, but from the lower carbamoyl-phosphate synthetase activity of the mutant mitochondria. (4) At lower external [ornithine] (less than 2 mM), a smaller fraction of the carbamoyl phosphate synthesized is converted into citrulline in spfash than in normal mitochondria. These studies show that what appears to be a single mutation brings about major adaptations in the mitochondrial component of liver. In addition, they clarify the role of ornithine transport and of protein-protein interactions in citrulline synthesis in normal mitochondria.     

Sequence of a tryptic peptide from the NADPH binding site of the enoyl reductase domain of fatty acid synthase

Fatty acid synthase from the uropygial gland of goose was inhibited by treatment with pyridoxal 5'-phosphate by selectively modifying a lysine residue at the NADPH binding site of the enoyl reductase domain (A. J. Poulose and P. E. Kolattukudy (1980) Arch. Biochem. Biophys. 201, 313-321). Distribution of radioactivity in tryptic peptides generated from the synthase treated with pyridoxal 5'-phosphate/NaB3H4 in the presence and absence of 2'-monophosphoadenosine-5'-diphosphoribose, which protects the enzyme from inactivation by pyridoxal phosphate, showed that modification of one specific peptide was prevented by the protector. This peptide was purified by a combination of Sephadex G-25 column chromatography, anion-exchange chromatography, and high-performance liquid chromatography. The primary structure of this peptide is Val-Phe-Thr-Thr-Val-Gly-Ser-Ala-Glu-Lys(Pxy)-Arg.     

Purification and characterization of NADP-specific alcohol dehydrogenase and glutamate dehydrogenase from the hyperthermophilic archaeon Thermococcus litoralis.

Thermococcus litoralis is a strictly anaerobic archaeon that grows at temperatures up to 98 degrees C by fermenting peptides. Little is known about the primary metabolic pathways of this organism and, in particular, the role of enzymes that are dependent on thermolabile nicotinamide nucleotides. In this paper we show that the cytoplasmic fraction of cell extracts contained NADP-specific glutamate dehydrogenase (GDH) and NADP-specific alcohol dehydrogenase (ADH) activities, neither of which utilized NAD as a cofactor. The GDH is composed of identical subunits having an M(r) of 45,000 and had an optimal pH and optimal temperature for glutamate oxidation of 8.0 and > 95 degrees C, respectively. Potassium phosphate (60 mM), KCl (300 mM), and NaCl (300 mM) each stimulated the rate of glutamate oxidation activity between two- and threefold. For glutamate oxidation the apparent Km values at 80 degrees C for glutamate and NADP were 0.22 and 0.029 mM, respectively, and for 2-ketoglutarate reduction the apparent Km values for 2-ketoglutarate, NADPH, and NH4+ were 0.16, 0.14, and 0.63 mM, respectively. This enzyme is the first NADP-specific GDH purified form a hyperthermophilic organism. T. litoralis ADH is a tetrameric protein composed of identical subunits having an M(r) of 48,000; the optimal pH and optimal temperature for ethanol oxidation were 8.8 and 80 degrees C, respectively. In contrast to GDH activity, potassium phosphate (60 mM), KCl (0.1 M), and NaCl (0.3 M) inhibited ADH activity, whereas (NH4)2SO4 (0.1 M) had a slight stimulating effect. This enzyme exhibited broad substrate specificity for primary alcohols, but secondary alcohols were not oxidized.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of aspartate and aspartate-derived antimetabolites with the enzymes of the threonine biosynthetic pathway of Escherichia coli

The five enzymes responsible for the conversion of L-aspartate to L-threonine in Escherichia coli were purified to homogeneity and subsequently reconstituted in vitro in ratios approximating those found in vivo. 31P NMR was used to conveniently monitor the rates of consumption of the substrates ATP and NADPH, the accumulation of the intermediates beta-aspartyl phosphate and homoserine phosphate, and the formation of the products ADP, NADP+, and Pi in a single experiment. By this method, the flux of aspartic acid through the enzymes of the pathway was monitored in the absence and in the presence of several alternative substrates and inhibitors. Several known antimetabolites were found to be alternative substrates that ultimately became inhibitors of pathway flux. L-threo-3-Hydroxyaspartic acid was converted to 3-hydroxyhomoserine phosphate by the first four enzymes of the pathway. The antimetabolite L-threo-3-hydroxyhomoserine was found to bind to and inhibit aspartokinase-homoserine dehydrogenase I in a cooperative fashion (I 0.5 = 3 mM, nH = 2.5), similar to the action of the allosteric end product inhibitor L-threonine (I 0.5 = 0.36 mM, nH = 2.4). In the presence of the remaining enzymes of the pathway, however, L-threo-3-hydroxyhomoserine was phosphorylated to the apparent ultimate antimetabolite L-threo-3-hydroxyhomoserine phosphate that was a potent inhibitor of threonine synthase and consequently of L-threonine biosynthesis. When aspartic acid alone was examined as a substrate of the enzymes of the pathway, no accumulation of the beta-aspartyl phosphate and homoserine phosphate intermediates was observed. However, in the presence of either 5 mM L-threo-3-hydroxyhomoserine or 5 mM L-threo-3-hydroxyhomoserine phosphate, homoserine phosphate was found to accumulate. In contrast to the homoserine phosphate and 3-hydroxyhomoserine phosphate intermediates, both of which were very stable, the acylphosphate intermediates beta-aspartyl phosphate and beta-3-hydroxyaspartyl phosphate were highly susceptible to hydrolysis, with first-order rate constants of 4.6 X 10(-3) min-1 and 4.5 X 10(-2) min-1 (pH 7.8, 25 degrees C), respectively.     

Effect of potassium ion on the phosphorus-31 nuclear magnetic resonance spectrum of the pyridoxal 5'-phosphate cofactor of Escherichia coli D-serine dehydratase

31P NMR studies were undertaken to determine how potassium ion increases the cofactor affinity of Escherichia coli D-serine dehydratase, a model pyridoxal 5'-phosphate requiring enzyme that converts the growth inhibitor D-serine to pyruvate and ammonia. Potassium ion was shown to promote the appearance of a second upfield shifted cofactor 31P resonance at 4.0 ppm (pH 7.8, 25 degrees C), that increased in area at the expense of the resonance at 4.4 ppm observed in the absence of K+. Na+ antagonized the K+ promoted appearance of the second resonance. These observations suggest that K+ and Na+ stabilize conformational states that differ with respect to O-P-O bond angle, conformation, and/or hydrogen bonding of the phosphate group. An analysis of the dependence of the relative intensities of the two resonances on the K+ concentration yielded a value of ca. 10 mM for the equilibrium constant for dissociation of K+ from D-serine dehydratase. The chemical shift difference between the two resonances indicated that the K+-stabilized and Na+-stabilized forms of the enzyme interconvert at a frequency less than 16 s-1 at pH 7.8, 25 degrees C.     

Aspartate aminotransferase ofLactobacillus murinus

Aspartate aminotransferase from Lactobacillus murinus is thermostable, its activity being not changed for two months at temperatures between 4 and -70 degrees C. Maximum activity was observed at 40 degrees C and pH 7.3 in phosphate buffer (30 mmol/L). delta G* Value of 26.3 kJ/mol was calculated from the Arrhenius plot. The Km values for L-aspartate and 2-oxoglutarate at pH 7.3 were 25 and 100 mmol/L, respectively. Sodium maleate and glutamate acted as inhibitors of the enzyme activity. The Ki values for sodium maleate with L-aspartate of 2-oxoglutarate as variable substrates were 1.1 and 0.5 mmol/L, respectively. The Ki values for glutamate with L-aspartate or 2-oxoglutarate were 8.0 and 4.0 mmol/L, respectively. An inhibitory effect was observed with 1 mM Hg2+ ions (1 mmol/L). The activity of the enzyme was diminished by only 12% in the absence of pyridoxal 5'-phosphate.     

Genetic analysis, nucleotide sequence, and products of two Pseudomonas denitrificans cob genes encoding nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase and cobalamin (5''-phosphate) synthase.

Tn5 Sp(r) transposons have been inserted into the 8-kb Pseudomonas denitrificans DNA fragment from complementation group D, which carries cob genes. Genetic analysis and the nucleotide sequence revealed that only two cob genes (cobU and cobV) were found on this cob genomic locus. Nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase (EC 2.4.2.21) was assayed and purified to homogeneity from a P. denitrificans strain in which cobU and cobV were amplified. The purified enzyme was identified as the cobU gene product on the basis of identical molecular weights and N-terminal sequences. Cobalamin (5'-phosphate) synthase activity was increased when cobV was amplified in P. denitrificans. The partially purified enzyme catalyzed not only the synthesis of cobalamin 5'-phosphate from GDP-cobinamide and alpha-ribazole 5'-phosphate but also the one-step synthesis of cobalamin from GDP-cobinamide and alpha-ribazole. Biochemical data provided evidence that cobV encodes cobalamin (5'-phosphate) synthase.     

Carbamoyl-phosphate synthetase: an example of effects on enzyme properties of shifting an equilibrium between active monomer and active oligomer

Carbamoyl-phosphate synthetase from Escherichia coli is subject to allosteric activation by ornithine, allosteric inhibition by uridine 5'-phosphate (UMP), and reversible concentration-dependent self-association. Positive allosteric effectors, magnesium adenosine 5'-triphosphate (MgATP), K+, and inorganic phosphate facilitate association. The purpose of this study was to determine the state of association of carbamoyl-phosphate synthetase in the presence and absence of different substrates and effectors and to consider the basis for the observed effects of enzyme concentration on specific activity. Studies employing gel filtration chromatography have shown that when the concentration of carbamoyl-phosphate synthetase is low (less than 0.01 mg/mL), the enzyme exists as monomer under all conditions, including the presence of UMP in phosphate buffer and the presence of all substrates plus ornithine (conditions that support maximal catalytic activity). At higher enzyme concentrations (e.g., greater than 0.01 mg/mL) the specific activity increases with increasing enzyme concentration when MgATP is nonsaturating but is independent of enzyme concentration when MgATP is saturating or when ornithine is present with MgATP being either saturating or nonsaturating. These results indicate that the catalytic activity of this enzyme is not directly linked to oligomer formation. The theoretical properties and possible significance of a generalized model of enzyme association-dissociation in which the active monomeric form, in equilibrium with another monomeric form, is specifically subject to self-association but the different states of association have the same specific activity, are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory properties of brain glutamate decarboxylase

1. Glutamate decarboxylase is a focal point for controlling gamma-aminobutyric acid (GABA) synthesis in brain. Several factors that appear to be important in the regulation of GABA synthesis have been identified by relating studies of purified glutamate decarboxylase to conditions in vivo. 2. The interaction of glutamate decarboxylase with its cofactor, pyridoxal 5'-phosphate, is a regulated process and appears to be one of the major means of controlling enzyme activity. The enzyme is present in brain predominantly as apoenzyme (inactive enzyme without bound cofactor). Studies with purified enzyme indicate that the relative amounts of apo- and holoenzyme are determined by the balance in a cycle that continuously interconverts the two. 3. The cycle that interconverts apo- and holoenzyme is part of the normal catalytic mechanism of the enzyme and is strongly affected by several probable regulatory compounds including pyridoxal 5'-phosphate, ATP, inorganic phosphate, and the amino acids glutamate, GABA, and aspartate. ATP and the amino acids promote apoenzyme formation and pyridoxal 5'-phosphate and inorganic phosphate promote holoenzyme formation. 4. Numerous studies indicate that brain contains multiple molecular forms of glutamate decarboxylase. Multiple forms that differ markedly in kinetic properties including their interactions with the cofactor have been isolated and characterized. The kinetic differences among the forms suggest that they play a significant role in the regulation of GABA synthesis.