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PubMed1977

Stereospecificity of phenylpyruvate tautomerase. A convenient method for the preparation of chirally labelled phenylpyruvates.

1. By 1H nuclear magnetic resonance spectroscopy it has been shown that phenylpyruvate tautomerase from beef kidney catalyses the stereospecific exchange of one of the enantiotopic 3H-atoms in the side-chain of the substrate with solvent protons, the rate of spontaneous exchange being slow under physiological conditions. 2. Using monodeuterated phenylpyruvate of known absolute configuration it has been shown that the tautomerase removes specifically the 3-pro-R hydrogen atom of the substrate. 3. The circular dichroism spectra of the two enantiomeric 3-phenyl-[3-2H]pyruvates have been determined. 4. Some implications of these findings for the investigation of the metabolism of aromatic amino acids are discussed.

Circular Dichroism↗
PubMed1987

Novel phenylalanine dehydrogenases from Sporosarcina ureae and Bacillus sphaericus. Purification and characterization.

NAD+-dependent phenylalanine dehydrogenases were purified 1,500- and 1,600-fold, and crystallized from Sporosarcina ureae SCRC-R04 and Bacillus sphaericus SCRC-R79a, respectively. The purified enzymes were homogeneous as judged by disc gel electrophoresis. The enzyme from S. ureae has a molecular weight of 305,000, while that of B. sphaericus has a molecular weight of 340,000. Each is probably composed of eight subunits identical in molecular weight. The S. ureae enzyme showed a high substrate specificity in the oxidative deamination reaction acting on L-phenylalanine, while that of B. sphaericus acted on L-phenylalanine and L-tyrosine. The enzymes had lower substrate specificities in the reductive amination reaction acting on alpha-keto acids. The Sporosarcina enzyme acted on phenylpyruvate, alpha-ketocaproate, alpha-keto-gamma-methylthiobutyrate and rho-hydroxyphenylpyruvate. The Bacillus enzyme acted on rho-hydroxyphenylpyruvate, phenylpyruvate, and alpha-keto-gamma-methylthiobutyrate. The enzyme from B. sphaericus catalyzes The enzyme from B. sphaericus catalyzes the transfer of pro-S (B) hydrogen from NADH.

Amino Acid Oxidoreductases↗
PubMed1984

Potent inhibitory effects of tyrosine metabolites on dihydropteridine reductase from human and sheep liver.

L-Phenylalanine and its metabolites, such as phenylpyruvate, phenylacetate and L-phenyllactate, do not significantly inhibit dihydropteridine reductase purified from human and sheep liver (I50 greater than or equal to 5 mM). However, L-tyrosine and its metabolites, such as L-DOPA, tyramine, p-hydroxyphenylpyruvate, p-hydroxyphenylacetate, and p-hydroxyphenylacetate, are potent noncompetitive inhibitors of this enzyme, with Ki values in the range 4-260 microM. These results suggest that tyrosine metabolites can potentially regulate levels of tetrahydrobiopterin, the required cofactor for the hydroxylation of aromatic amino acids.

Animals↗
PubMed1997

The macrophage migration inhibitory factor MIF is a phenylpyruvate tautomerase.

A macrophage migration inhibitory factor (MIF), originally described as a product of activated lymphocytes, has been defined as a 12 kDa protein, expressed in a wide variety of tissues. Here MIF is identified as a phenylpyruvate tautomerase (EC 5.3.2.1) having p-hydroxyphenylpyruvate and phenylpyruvate as its natural substrates. The definition of MIF as an enzyme may yield insight into the mechanism of action of this proinflammatory and immunomodulating cytokine.

Amino Acids↗
PubMed1972

The oxidation of Schiff bases of pyridoxamine with -oxo acids by manganous ions and peroxidase.

1. Oxygen was taken up rapidly when pyruvate was added to mixtures of pyridoxamine and Mn(2+) ions after lag periods that were shortened by peroxidase (donor-hydrogen peroxide oxidoreductase, EC 1.11.1.7). 2. The total oxygen uptake was proportional to the pyridoxamine added and was accompanied by the disappearance of pyridoxamine; the pyruvate acted catalytically and hydrogen peroxide was not formed. 3. At pH6 more than half the pyridoxamine that disappeared was accounted for as pyridoxal and ammonia; it is suggested that the primary reaction is the oxidative deamination of the pyridoxamine. 4. Results were similar when alpha-oxobutyrate or glyoxylate were substituted for pyruvate, except that the reactions were slower and the yield of pyridoxal less. 5. The oxidative decarboxylations of alpha-oxoglutarate and phenylpyruvate are catalysed by Mn(2+) ions and these reactions are activated by peroxidase; pyridoxamine increased both the rates and total oxygen uptakes in these reactions, and ammonia was produced. 6. The lag periods in the oxidation of mixtures of pyridoxamine and alpha-oxo acids, catalysed by Mn(2+) ions, were also shortened by traces of colloidal manganese dioxide. 7. It is suggested that the activating effect of peroxidase depends on its catalysis of manganese oxidation.

Ammonia↗
PubMed1974

Mis-regulation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase does not account for growth inhibition by phenylalanine in Agmenellum quadruplicatum.

The growth of the blue-green bacterium, Agmenellum quadruplicatum, is inhibited in the presence of l-phenylalanine. This species has a single, constitutively synthesized 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase. l-Phenylalanine inhibits DAHP synthetase non-competitively with respect to both substrate reactants. Other aromatic amino acids do not inhibit the activity of DAHP synthetase. A common expectation for branch-point enzymes such as DAHP synthetase is a balanced pattern of feedback control by all of the ultimate end products. It seemed likely that growth inhibition might equate with defective regulation within the branched aromatic pathway. Accordingly, the possibility was examined that mis-regulation of DAHP synthetase by l-phenylalanine in wild-type cells causes starvation for precursors of the other aromatic end products. However, the molecular basis for growth inhibition cannot be attributed to l-phenylalanine inhibition of DAHP synthetase for the following reasons: (i) DAHP synthetase enzymes from l-phenylalanine-resistant mutants are more, rather than less, sensitive to feedback inhibition by l-phenylalanine. (ii) Shikimate not only fails to antagonize inhibition, but is itself inhibitory. (iii) Neither the sensitivity nor the completeness of l-phenylalanine inhibition of the wild-type enzyme in vitro appears sufficient to account for the potent inhibition of growth in vivo by l-phenylalanine. The dominating effect of l-phenylalanine in the control of DAHP synthetase appears to reflect a mechanism that prevents rather than causes growth inhibition by l-phenylalanine. The alteration of the control of DAHP synthetase in mutants selected for resistance to growth inhibition by l-phenylalanine did indicate that the cause for this metabolite vulnerability can be localized within the aromatic amino acid pathway. Apparently, an aromatic intermediate (between shikimate and the end products) accumulates in the presence of l-phenylalanine, causing toxicity by some unknown mechanism. It is concluded that phenylpyruvate, potentially formed by transamination of l-phenylalanine, is an unlikely cause of growth inhibition. Although several significant questions remain unanswered, our results suggest that single-effector control of DAHP synthetase, the first regulatory enzyme activity of a branched pathway, may be more appropriate than it would seem a priori.

Amino Acid Isomerases↗
PubMed1977

Identity of rat kidney histidine-pyruvate aminotransferase with glutamine-oxo acid aminotransferase.

Histidine-pyruvate aminotransferase and glutamine-phenylpyruvate aminotransferase were purified from rat kidney by the same procedure. The ratio of the two activities remained constant during purification and was unchanged by a variety of treatments of the purified enzyme. Glutamine was found to act as a competitive inhibitor of histidine-pyruvate aminotransferase. These results suggest that rat kidney histidine-pyruvate aminotransferase is identical with glutamine-oxo acid aminotransferase. Identity of the two enzymes in other tissues of rats is discussed.

Animals↗
PubMed1966

Distribution and function of genes concerned with aromatic biosynthesis in Escherichia coli.

Pittard, James (School of Microbiology, University of Melbourne, Victoria, Australia), and B. J. Wallace. Distribution and function of genes concerned with aromatic biosynthesis in Escherichia coli. J. Bacteriol. 91:1494-1508. 1966.-A number of mutant strains of Escherichia coli K-12, which are blocked in the biosynthesis of the aromatic amino acids, were examined biochemically to determine their particular enzymatic deficiencies. The mutations carried by these strains were mapped by use of the methods of conjugation and transduction. Structural genes for five of the enzymes of the common pathway leading to chorismate and for the two enzymes converting chorismate to phenylpyruvate and p-hydroxyphenylpyruvate, respectively, were identified. Unlike the genes of the tryptophan operon most of these genes are distributed over widely separated regions of the chromosome.

Amino Acids↗
PubMed1973

Channel-shuttle mechanism for the regulation of phenylalanine and tyrosine synthesis at a metabolic branch point in Pseudomonas aeruginosa.

A bifunctional protein complex was partially purified from Pseudomonas aeruginosa. Catalytic activities for chorismate mutase and prephenate dehydratase coeluted from gel filtration and DEAE-cellulose chromatography columns. The protein complex had a molecular weight of approximately 134,000, as determined by gel filtration. In crude extracts or in partially purified preparations about one-half of the chorismate utilized by the complex is converted to phenylpyruvate, and the other half accumulates as prephenate. The chorismate mutase activity is strongly product-inhibited by prephenate, competitively with chorismate. Accordingly, the first reaction of the complex can be sufficiently retarded by prephenate so that all of the reaction product is phenylpyruvate. Chorismate mutase activity is also competitively inhibited by phenylalanine. Although phenylalanine is effective at low concentrations, maximal inhibition is only 50 to 60%. Inhibition of chorismate mutase by phenylalanine was completely lost after gel filtration. The prephenate dehydratase activity of the protein complex is nearly completely inhibited by 0.1 mm phenylalanine in either crude extracts or partially purified preparations. A second species of prephenate dehydratase was separated from the prephenate dehydratase-chorismate mutase aggregate by gel filtration or anion exchange chromatography. The second prephenate dehydratase had an estimated molecular weight of 76,000, a high affinity for prephenate, and was insensitive to feedback inhibition by phenylalanine. The physiological role of the latter enzyme is uncertain. The other regulatory enzymes of tyrosine and phenylalanine biosynthesis, prephenate mutase aggregate by gel filtration or anion exchange chromatography. The other regulatory enzymes of tyrosine and phenylalanine biosynthesis, prephenate dehydrogenase (molecular weight of 120,000) and 3-deoxy-d-arabino-heptulosonate-7-phosphate synthetase (molecular weight of 52,000), elute from Sephadex G-100 columns as fractions which are distinct from both the chorismate mutase-prephenate dehydratase complex and from the low-molecular-weight species of prephenate dehydratase. A shuttle mechanism governing the metabolic fate of prephenate (to phenylalanine or to tyrosine) is proposed in the context of a model which also accommodates several previously puzzling findings: (i) the dominating role of tyrosine in the control of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthetase and (ii) the lack of feedback control of prephenate dehydrogenase by tyrosine.

Aldehyde-Lyases↗
PubMed2003

(4-Hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis: the basis for ordered substrate addition.

(4-hydroxyphenyl)pyruvate dioxygenase (HPPD) catalyzes the second step in the pathway for the catabolism of tyrosine, the conversion of (4-hydroxyphenyl)pyruvate (HPP) to homogentisate (HG). This reaction involves decarboxylation, substituent migration, and aromatic oxygenation. HPPD is a member of the alpha-keto acid dependent oxygenases that require Fe(II) and an alpha-keto acid substrate to oxygenate an organic molecule. We have examined the binding of ligands to HPPD from Streptomyces avermitilis. Our data show that HPP binds to the apoenzyme and that the apo-HPPD.HPP complex does not bind Fe(II) to generate active holoenzyme. The binding of HPP, phenylpyruvate (PPA), and pyruvate to the holoenzyme produces a weak ligand charge-transfer band at approximately 500 nm that is indicative of bidentate binding of the 1-carboxylate and 2-keto pyruvate oxygen atoms to the active site metal ion. For HPPD from this organism the 4-hydroxyl group of (4-hydroxyphenyl)pyruvate is a requirement for catalysis; no turnover is observed in the presence of phenylpyruvate. The rate constant for the dissociation of Fe(II) from the holoenzyme is 0.0006 s(-)(1) and indicates that this phenomenon is not significantly relevant in steady-state turnover. The addition of HPP and molecular oxygen to the holoenzyme is formally random. The basis of the ordered bi bi steady-state kinetic mechanism previously observed by Rundgren (Rundgren, M. (1977) J. Biol. Chem. 252, 5094-9) is the 3600-fold increase in oxygen reactivity when holo-HPPD is in complex with HPP. This complex reacts with molecular oxygen with a second-order rate constant of 1.4 x 10(5) M(-)(1) s(-)(1) inducing the formation of an intermediate that decays at the catalytically relevant rate of 7.8 s(-)(1).

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed1998

Mitochondrial uncoupling protein may participate in futile cycling of pyruvate and other monocarboxylates.

The physiological role of monocarboxylate transport in brown adipose tissue mitochondria has been reevaluated. We studied pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and phenylpyruvate uniport via the uncoupling protein (UCP1) as a GDP-sensitive swelling in K+ salts induced by valinomycin or by monensin and carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone in Na+ salts. We have demonstrated that this uniport is inhibited by fatty acids. GDP inhibition in K+ salts was not abolished by an uncoupler, indicating a negligible monocarboxylic acid penetration via the lipid bilayer. In contrast, the electroneutral pyruvate uptake (swelling in ammonium pyruvate or potassium pyruvate induced by change in pH) mediated by the pyruvate carrier was inhibited by its specific inhibitor alpha-cyano-4-hydroxycinnamate but not by fatty acids. Moreover, alpha-cyano-4-hydroxycinnamate enhanced the energization of brown adipose tissue mitochondria, which was monitored fluorometrically by 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide and safranin O. Consequently, we suggest that UCP1 might participate in futile cycling of unipolar ketocarboxylates under certain physiological conditions while expelling these anions from the matrix. The cycle is completed on their return via the pyruvate carrier in an H+ symport mode.

Adipose Tissue, Brown↗
PubMed1982

The stereochemical course of the water elimination from (2R)-phenyllactate in the amino acid fermentation of Clostridium sporogenes.

(2R,3R)-[3-3H]- and (2R,3S)-[3-3H]- phenyllactate were synthesized from phenylpyruvate with the help of phenylpyruvate tautomerase and an NADH-dependent 2-oxo-acid reductase from Clostridium sporogenes. With whole cells of C. sporogenes both (2R)-phenyllactates are mainly transformed to phenylpropionate and, to a minor extent to phenylalanine. According to recently published results, the former transformation leads from phenyllactate, or an activated derivative of it, to (E)-cinnamate which is reduced to phenylpropionate in a fast consecutive reaction. The 3H/14C-ratio of phenylpropionate deriving from (2R,3R)-[3-3H,U-14C]phenyllactate was about 85% of that of the phenyllactate and the corresponding ratio for phenylpropionate deriving from the (2R,3S)-[3-3H,U-14C]phenyllactate about 7%, respectively. Assuming that the E-configuration of cinnamate is formed directly from (2R)-phenyllactate, the water elimination occurs in a syn fashion.

Amino Acids↗
PubMed1983

Mechanism of 3-phenylpyruvate-induced insulin release. Secretory, ionic and oxidative aspects.

1. 3-Phenylpyruvate caused a dose-related stimulation of insulin release from rat pancreatic islets deprived of exogenous nutrient or incubated in the presence of 5.6 or 8.3 mM-D-glucose. 2. 3-Phenylpyruvate inhibited insulin release evoked by high concentrations of D-glucose (16.7 or 27.8 mM) or 4-methyl-2-oxopentanoate (10.0 mM). This inhibitory effect appeared to be attributable to impairment of 2-oxo-acid transport into the mitochondria, with resulting inhibition of D-glucose, pyruvate or 4-methyl-2-oxopentanoate oxidation. 3. 3-Phenylpyruvate failed to affect the oxidation of, and secretory response to, L-leucine, and did not augment insulin release evoked by a non-metabolized analogue of the latter amino acid. 4. L-Glutamine augmented 3-phenylpyruvate-induced insulin release. The release of insulin evoked by the combination of 3-phenylpyruvate and L-glutamine represented a sustained phenomenon, abolished in the absence of extracellular Ca2+ or the presence of menadione and potentiated by theophylline. 5. Whether in the presence or in the absence of L-glutamine, the secretory response to 3-phenylpyruvate coincided with an increase in O2 uptake, a decrease in K+ conductance, a stimulation of both Ca2+ inflow and 45Ca2+ net uptake and an increase in cyclic AMP content. 6. It is concluded that the release of insulin induced by 3-phenylpyruvate displays features classically encountered when the B-cell is stimulated by nutrient secretagogues, and is indeed attributable to an increase in nutrient catabolism.

Animals↗
PubMed1982

A multispecific quintet of aromatic aminotransferases that overlap different biochemical pathways in Pseudomonas aeruginosa.

Pseudomonas aeruginosa possesses dual enzymatic sequences to both L-phenylalanine and L-tyrosine, a biosynthetic arrangement further complicated by the presence of five aromatic aminotransferases. Each aminotransferase is capable of transamination in vitro with any of the three keto acid intermediates in the aromatic pathway (phenylpyruvate, 4-hydroxyphenylpyruvate, or prephenate). The fractional contribution of these aminotransferases to particular transamination reactions in vivo can best be approached through the systematic and sequential elimination of individual aminotransferase activities by mutation. A program of sequential mutagenesis has produced two aminotransferase-deficient mutations. The first mutation imposed a phenotype of bradytrophy for L-phenylalanine (doubling time of 2.4 h in minimal salts/glucose medium compared to a 1.0-h doubling time for wild type). This mutant completely lacked an enzyme denoted aminotransferase AT-2. A genetic background of aminotransferase AT-2 deficiency was used to select for a second mutation which produced a phenotype of multiple auxotrophy for L-phenylalanine, L-aspartate, and L-glutamate. The double mutant completely lacked activity for aromatic aminotransferase AT-1 in addition to the missing aminotransferase AT-2. Enzymes AT-1 (Mr = 64,000) and AT-2 (Mr = 50,000) were readily separated from one another by gel filtration and were individually characterized for pH optima, freeze-thaw stability, heat lability, and molecular weight. The phenotypic and enzymological characterizations of the aminotransferase mutants strongly support the primary in vivo role of enzyme AT-2 in L-phenylalanine and L-tyrosine biosynthesis, while enzyme AT-1 must primarily be engaged in L-aspartate and L-glutamate synthesis. The substrate specificities and possible in vivo functions for AT-3, AT-4, and AT-5 are also considered.

Aspartate Aminotransferases↗
PubMed1971

Enzyme alterations in tyrosine and phenylalanine auxotrophs of Salmonella typhimurium.

The enzyme activities specified by the tyrA and pheA genes were studied in wildtype strain Salmonella typhimurium and in phenylalanine and tyrosine auxotrophs. As in Aerobacter aerogenes and Escherichia coli, the wild-type enzymes of Salmonella catalyze two consecutive reactions: chorismate --> prephenate --> 4-hydroxy-phenylpyruvate (tyrA), and chorismate --> prephenate --> phenylpyruvate (pheA). A group of tyrA mutants capable of interallelic complementation had altered enzymes which retained chorismate mutase T activity but lacked prephenate dehydrogenase. Similarly, pheA mutants (in which interallelic complementation does not occur) had one group with altered enzymes which retained chorismate mutase P but lacked prephenate dehydratase. Tyrosine and phenylalanine auxotrophs outside of these categories showed loss of both activities of their respective bifunctional enzyme. TyrA mutants which had mutase T were considerably derepressed in this activity by tyrosine starvation and consequently excreted prephenate. A new and specific procedure was developed for assaying prephenate dehydrogenase activity.

Cell-Free System↗
PubMed1978

Myelin deficiency in experimental phenylketonuria: contribution of the aromatic acid metabolites of phenylalanine.

Retarded body and brain growth and a deficit of myelin in the cerebral hemispheres and the cerebellum were observed in an animal model of phenylketonuria, the p-chlorophenylalanine and L-phenylalanine treated preweanling rat. These manifestations of phenylketonuria were reproduced in rats treated with phenylacetate in amounts approximating those likely to be produced in phenylketonuria. Young rats treated with equivalent amounts of other metabolites of phenylalanine, namely, phenylpyruvate, phenyllactate, and mandelate, which also accumulate in the brain during hyperphenylalaninemia, did not exhibit any toxic effects. Phenylpyruvate did not give rise to phenylacetate in the brain, but a small percentage was converted to phenyllactate. The gross composition of myelin isolated from the brains of saline and phenylacetate treated animals was similar. At various time intervals after subcutaneous injection, phenylacetate in the brain reached levels thirty times those of phenylpyruvate and phenyllactate, although animals received equivalent amounts of the three metabolites. The retarded growth of the body and brain of the young animal treated with phenylacetate may be attributed to the formation of phenylacetylcoenzyme A in the tissues. The site of action is very likely linked to acylcoenzyme A metabolism, i.e., the synthesis and utilization of acetylCoA and acetoacetylCoA, which are involved in reactions generating ATP and energy and in the synthesis of cholesterol and fatty acids. Results of this investigation indicate that growth retardation induced by phenylacetate during the period of very rapid development of the brain is responsible for the mental retardation in phenylketonuria.

Animals↗