TRANSAMINATIONS BETWEEN AMINO ACIDS AND KETO ACIDS ELEVATED IN PHENYLKETONURIA AND MAPLE SYRUP URINE DISEASE

Abstract— The transamination between amino acids and aliphatic and aromatic keto acids has been investigated in homogenates of human and rat brain. Tryptophan, phenylalanine and 3,4-dihydroxyphenylalanine (DOPA) at concentrations of 3.6 min and below trans-aminated aromatic keto acids more rapidly than α-ketoglutarate; lower K_m values were found for tryptophan and phenylalanine in the presence of the aromatic keto acid. Rat brain and liver arninotransferases exhibited similar affinities for tryptophan in the presence of different keto acids. Branched chain keto acids were also acceptors of the amino groups of tryptophan and DOPA. In brain homogenates α-ketoglutarate and p -hydroxyphenyl-pyruvate were transaminated by tyrosine and 5-hydroxytryptophan at about equal rates, whereas a-ketoglutarate was transaminated more rapidly with aliphatic amino acids. At concentrations of 1.6 m DOPA and 0.8 mM p -hydroxyphenylpyruvate, transamination was 6-fold greater than the rate of formation of dopamine. The dihydroxyphenylpyruvate formed during arninotransfer from DOPA by brain tissue was not readily decarboxylated, whereas 65–70 per cent of the indolepyruvate formed from tryptophan was decarboxylated. We suggest that an increased rate or degree of transamination between tryptophan and aromatic and branched chain keto acids may explain the increased excretion of non-hydroxylated indolic acids in phenylketonuria and'maple syrup urine'disease, respectively. Increased aminotransfers from tryptophan and DOPA may reduce the amounts of precursors available for the synthesis of serotonin and catecholamines, both of which are at low levels in the sera of untreated phenylketonurics.     

AN EXAMINATION OF THE CATALYTIC PROPERTIES OF SEVERAL AMINOTRANSFERASES IN BRAIN AND LIVER BY MEANS OF AN IMPROVED RADIOMETRIC ASSAY WITH DINITROPHENYLHYDRAZINE

Abstract— The assay of aminotransferases, performed by solvent extraction of keto acids formed from labelled amino acids, has been modified to enhance the recovery of both aliphatic and aromatic keto acid products. The keto acids are first converted to their respective dinitrophenylhydrazones which are more completely extracted into less polar organic solvents. By this manoeuvre, both keto acid extraction is increased and the extraction of the precursor amino acid is reduced. Employing this technique, the kinetics of brain-stem γ-aminobutyric acid (GABA), tryptophan, 3,4-dihydroxyphenylalanine (DOPA) aminotransferases and brain-stem and liver tyrosine aminotransferases were examined. Brain-stem aminotransferases, particularly the aromatic amino acid transferases, have a higher affinity for both the amino acid and the keto acid when the aromatic keto acid, phenylpyruvate (0·8 mM), is employed as amino group acceptor, whereas maximal velocities for aminotransferase reactions are much greater when α-ketoglutarate (0·8 m m ) is the amino group acceptor. Brain-stem tyrosine aminotransferase exhibits a much lower affinity for tyrosine in the presence of either 0·8m m -α-ketoglutarate or 0·8 m m -phenylpyruvate than does liver tyrosine aminotransferase. p -Chlorophenylpyruvate and phenylpyruvate exhibit similar properties as amino group acceptors for brain-stem tryptophan aminotransferase. Cysteine inhibits tryptophan aminotransferase when phenylpyruvate is the amino group acceptor, in a manner which is competitive with the amino acid. Benzoylformate inhibits both tryptophan and DOPA aminotransferases when phenylpyruvate is the amino group acceptor, but this inhibition does not appear to be competitive with phenylpyruvate.     

Differences in properties between aromatic amino acid: Aromatic keto acid aminotransferases and aromatic amino acid: α-ketoglutarate aminotransferases

Homogenates of rat liver transaminate phenylpyruvate (PP), as well as α-ketoglutarate (α-KG), in the presence of L-tyrosine, 3,4-dihydroxyphenylalanine (L-DOPA) or L-tryptophan. Aminotransferase activity with phenylpyruvate and DOPA, but not with tyrosine, was inhibited by excess phenylpyruvate. Tyrosine and DOPA aminotransferase activities with phenylpyruvate were more heat stable than the corresponding activities with α-ketoglutarate. Aminotransferase activities with phenylpyruvate were not significantly induced following intraperitoneal injections of cortisol, glucagon or serotonin, compared with a 3 to 7-fold increase in the aminotransferase activities with α-ketoglutarate. Tyrosine:phenylpyruvate aminotransferase activity rose 40% at night, compared with a 300% increase in tyrosine:α-ketoglutarate aminotransferase activity. The results suggest that aminotransferases catalysing transfers between aromatic keto acids and aromatic amino acids are separate enzymes from those utilizing α-ketoglutarate as the acceptor keto acid.     

TRANSAMINATION OF AMINO ACIDS IN HOMOGENATES OF RAT BRAIN

Abstract— The aminotransferase activity of homogenates of brains from adult and neonatal rats has been investigated. Aminotransferase activity was demonstrated wtih 15 of 22 amino acids incubated with seven keto acids. The basic amino acids exhibited little or no activity.

• 1 The greatest activity was obtained when glutamate or aspartate was incubated with α-ketoglutarate or oxaloacetate. Significant activity was also observed when the neutral aliphatic and aromatic amino acids were incubated with these two keto acids.
• 2 Activity with pyruvate was obtained principally upon incubation with glutamate and alanine. Most of the other amino acids that underwent transamination with α-ketoglutarate also did so with pyruvate, although at a lower rate.
• 3 When phenylpyruvate was added to the medium, glutamate, phenylalanine and tyrosine transaminated most actively.
• 4 Incubations with 11 amino acids and glyoxylic acid demonstrated aminotransferase activity, with glutamate and ornithine being the most active substrates.
• 5 α-Ketoisocaproate and α-ketoisovalerate accepted amino groups primarily from the branched-chain amino acids. Except for glutamate, activity with other amino acids was low or not detectable.
• 6 A comparison of aminotransferase activity in the newborn brain with that in the adult brain showed that the greatest change in activity occurred for glutamate with pyruvate or for alanine with α-ketoglutarate, these activities increasing about 10-fold from birth to adulthood; during this time activities with most other amino acids increased two- to threefold. Amino transfers from the branched-chain amino acids showed no increase with maturation, and some reactions, such as that with methionine and a number of keto acids, decreased from birth to adulthood.
• 7 Our results correspond in general to previous studies of aminotransferase activity in brain and in liver. However, our study also indicates a possible second aminotransferase acting on the branched-chain amino acids, the presence of aminotransferase activity for methionine and asparagine, and relatively high aminotransferase activity for glutamine or ornithine when incubated with glyoxylic acid rather than other keto acids. Moreover, phenylpyruvate and glyoxylate are active in amino transfers and may serve as substrates for a number of aminotransferases.

     

Tryptophan degradation in Saccharomyces cerevisiae: Characterization of two aromatic aminotransferases

Tryptophan was found to be degraded in Saccharomyces cerevisiae mainly to tryptophol. Upon chromatography on DEAE-cellulose two aminotransferases were identified: Aromatic aminotransferase I was constitutively synthesized and was active in vitro with tryptophan, phenylalanine or tyrosine as amino donors and pyruvate, phenylpyruvate or 2-oxoglutarate as amino acceptors. The enzyme was six times less active with and had a twenty times lower affinity for tryptophan (K _m=6 mM) than phenylalanine or tyrosine. It was postulated thus that aromatic aminotransferase I is involved in vivo in the last step of tyrosine and phenylalanine biosynthesis. Aromatic aminotransferase II was inducible with tryptophan but also with the other two aromatic amino acids either alone or in combinations. With tryptophan as amino donor the enzyme was most active with phenylpyruvate and not active with 2-oxoglutarate as amino acceptor; its affinity for tryptophan was similar as for the other aromatic amino acids (K _m=0.2–0.4 mM). Aromatic aminotransferase II was postulated to be involved in vivo mainly in the degradation of tryptophan, but may play also a role in the degradation of the other aromatic amino acids.A mutant strain defective in the aromatic aminotransferase II (aat2) was isolated and its influence on tryptophan accumulation and pool was studied. In combination with mutations trp2 ^fbr, aro7 and cdr1-1, mutation aat2 led to a threefold increase of the tryptophan pool as compared to a strain with an intact aromatic aminotransferase II.     

Transamination of neutral amino acids and 2-keto acids in pancreatic B-cell mitochondria

High aminotransferase activities catalyzing the reactions between L-glutamate and L-glutamine and the aliphatic ketomonocarboxylic acids 2-ketoisocaproate, 2-ketocaproate, and 2-ketoisovalerate were observed in pancreatic B-cell mitochondria. While maximal rates of transamination with L-glutamate were observed in the presence of micromolar concentrations of keto acid, maximal rates of transamination with L-glutamine were recorded only in the presence of millimolar concentrations of keto acid. The insulin secretagogue 2-ketoisocaproate was the most effective transamination partner for L-glutamate, while the insulin secretagogue 2-ketocaproate was the most effective transamination partner for L-glutamine. Since B-cell mitochondria are well supplied with L-glutamate and L-glutamine, 2-ketoglutarate generation in the presence of these two neutral 2-keto acids may be an important prerequisite for their insulin secretory potency. High rates of transamination of 2-ketoglutarate were observed in the pancreatic B-cell mitochondria with the branched-chain amino acids L-leucine and L-valine, but not with L-norleucine. In connection with the ability of L-leucine to activate glutamate dehydrogenase, this high activity of the branched-chain amino acid aminotransferase in pancreatic B-cell mitochondria may provide an explanation for the insulin secretory potency of this amino acid.     

AROMATIC AMINOTRANSFERASE ACTIVITY OF RAT BRAIN CYTOPLASMIC AND MITOCHONDRIAL ASPARTATE AMINOTRANSFERASES

Abstract— Mitochondrial and cytoplasmic forms of aspartate aminotransferase were purified from rat brain homogenates and tested for their ability to catalyze transamination of various aromatic amino acids. The mitochondrial enzyme exhibited activity toward tyrosine and phenylalanine with 2-oxoglutar-ate as acceptor, although the specific activities were less than 1% of the corresponding aspartate activity when all substrates were 10 mM. Even less activity was seen with DOPA, 5-hydroxytryptophan and tryptophan. The cytoplasmic aspartate aminotransferase was active toward tryptophan, 5-hydroxytryptophan and DOPA, but these transaminations were favored by pyruvate or oxaloacetate rather than 2-oxoglutarate as keto acid. Based on co-migration of aromatic activities with the respective aspartate aminotransferases during isoelectric focusing and based on equal sensitivities of aromatic transamination and aspartate transamination to inhibition by vinylglycine, it was concluded that all activities resided in the aspartate aminotransferase enzymes. Some doubt exists, however, as to the physiological significance of these alternate activities in view of the requirement that aromatic amino acids must compete with aspartate for transamination by these enzymes.     

Characterization of an aromatic amino acid aminotransferase from Rhizobium leguminosarum biovar trifolii.

The most abundant aromatic amino acid aminotransferase of Rhizobium leguminosarum biovar trifolii was partially purified. The molecular mass of the enzyme was estimated to be 53 kDa by gel filtration. The enzyme transaminated aromatic amino acids and histidine. It used aromatic keto acids and alpha-ketoglutaric and oxalacetic acids as amino-group acceptors. The optimum temperature was 35 degrees C. Using phenylalanine and alpha-ketoglutaric acid as substrates the activation energy was 46.2 kJ.mol-1 and for the couple tryptophan:alpha-ketoglutaric acid it was 70.3 kJ.mol-1. The optimum pH was different for each substrate: 7.3 for phenylalanine, 7.9 for histidine and 8.7 for tryptophan.     

The influence of starvation and tryptophan administration on the metabolism of phenylalanine, tyrosine and tryptophan in isolated rat liver cells.

Liver cells from fed Sprague-Dawley rats metabolized phenylalanine, tyrosine and tryptophan at rates consistent with the known kinetic properties of the first enzymes of each pathway. Starvation of rats for 48 h did not increase the maximal activities of phenylalanine hydroxylase, tryptophan 2,3-dioxygenase and tyrosine aminotransferase in liver cell extracts, when results were expressed in terms of cellular DNA. Catabolic flux through the first two enzymes was unchanged; that through the aminotransferase was elevated relatively to enzyme activity. This is interpreted in terms of changes in the concentrations of 2-oxoglutarate and glutamate. Cells from tryptophan-treated animals exhibited significant increases in the catabolism of tyrosine and tryptophan, but not of phenylalanine. The activities of tyrosine aminotransferase and tryptophan 2,3-dioxygenase were also increased, although the changes in flux and enzyme activity did not correspond exactly. These results are discussed with reference to the control of aromatic amino acid catabolism in liver; the role of substrate concentration is emphasized.     

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.     

Quantification of the importance of individual steps in the control of aromatic amino acid metabolism.

The quantitative importance of the individual steps of aromatic amino acid metabolism in rat liver was determined by calculation of the respective Control Coefficients (Strengths). The Control Coefficient of tryptophan 2,3-dioxygenase for tryptophan degradation was determined in a variety of physiological conditions and with a range of activities of tryptophan 2,3-dioxygenase. The Control Coefficient varied from 0.75 with basal enzyme activity to 0.25 after maximal induction of the enzyme by dexamethasone. The remainder of the control for tryptophan degradation was associated with the transport of the amino acid across the plasma membrane, with only very small contributions from kynureninase and kynurenine hydroxylase. The Control Coefficients of tyrosine aminotransferase for tyrosine degradation were approx. 0.70 and 0.20 with basal and dexamethasone-induced tyrosine aminotransferase activities respectively; the Control Coefficients of the transport of the amino acid into the cell were 0.22 and 0.58 respectively. Phenylalanine hydroxylase was found to have a Control Coefficient for the degradation of phenylalanine of approx. 0.50 under conditions of basal enzyme activity; after maximal activation by glucagon, the Control Coefficient decreased to 0.12. The transport of phenylalanine was responsible for the remaining control in the pathway. These results have important implications, directly for the regulation of aromatic amino acid metabolism in the liver, and indirectly for the regulation of neuroamine synthesis in the brain.     

Transamination of 3-phenylpyruvate in pancreatic B-cell mitochondria

High aminotransferase activities catalyzing the reaction between L-glutamate and the aromatic ketomonocarboxylic acid, 3-phenylpyruvate, were observed in the mitochondria from pancreatic B-cells. At very low concentrations of 3-phenylpyruvate, L-glutamine was an effective amino group donor. The aminotransferase activities for the aliphatic ketomonocarboxylic acids, pyruvate and 2-ketoisovalerate, were lower in B-cell mitochondria. High rates of transamination of 2-ketoisocaproate with L-glutamine were observed and may be an important prerequisite for the insulin secretory potency of this 2-keto acid. Since B-cell mitochondria are well supplied with L-glutamine and L-glutamate, 3-phenylpyruvate-induced 2-ketoglutarate production may explain the insulin secretory potency of 3-phenylpyruvate which is not a fuel for pancreatic islet cells.     

Rerouting Citrate Metabolism in Lactococcus lactis to Citrate-Driven Transamination

Oxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm of Lactococcus lactis ILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate by the citrate transporter CitP (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:4049-4056, 2011). In this study, transamination of amino acids with oxaloacetate as the keto donor is described as an additional mechanism to relieve toxic stress. Redirection of the citrate metabolic pathway into the transamination route in the presence of the branched-chain amino acids Ile, Leu, and Val; the aromatic amino acids Phe, Trp, and Tyr; and Met resulted in the formation of aspartate and the corresponding α-keto acids. Cells grown in the presence of citrate showed 3.5 to 7 times higher transaminase activity in the cytoplasm than cells grown in the absence of citrate. The study demonstrates that transaminases of L. lactis accept oxaloacetate as a keto donor. A significant fraction of 2-keto-4-methylthiobutyrate formed from methionine by citrate-driven transamination in vivo was further metabolized, yielding the cheese aroma compounds 2-hydroxy-4-methylthiobutyrate and methyl-3-methylthiopropionate. Reducing equivalents required for the former compound were produced in the citrate fermentation pathway as NADH. Similarly, phenylpyruvate, the transamination product of phenylalanine, was reduced to phenyllactate, while the dehydrogenase activity was not observed for the branched-chain keto acids. Both α-keto acids and α-hydroxy acids are known substrates of CitP and may be excreted from the cell in exchange for citrate or oxaloacetate.     

Role of mitochondrial transamination in branched chain amino acid metabolism

Oxidative decarboxylation and transamination of 1-14C-branched chain amino and alpha-keto acids were examined in mitochondria isolated from rat heart. Transamination was inhibited by aminooxyacetate, but not by L-cycloserine. At equimolar concentrations of alpha-ketoiso[1-14C]valerate (KIV) and isoleucine, transamination was increased by disrupting the mitochondria with detergent which suggests transport may be one factor affecting the rate of transamination. Next, the subcellular distribution of the aminotransferase(s) was determined. Branched chain aminotransferase activity was measured using two concentrations of isoleucine as amino donor and [1-14C]KIV as amino acceptor. The data show that branched chain aminotransferase activity is located exclusively in the mitochondria in rat heart. Metabolism of extramitochondrial branched chain alpha-keto acids was examined using 20 microM [1-14C]KIV and alpha-ketoiso[1-14C]caproate (KIC). There was rapid uptake and oxidation of labeled branched chain alpha-keto acid, and, regardless of the experimental condition, greater than 90% of the labeled keto acid substrate was metabolized during the 20-min incubation. When a branched chain amino acid (200 microM) or glutamate (5 mM) was present, 30-40% of the labeled keto acid was transaminated while the remainder was oxidized. Provision of an alternate amino acceptor in the form of alpha-keto-glutarate (0.5 mM) decreased transamination of the labeled KIV or KIC and increased oxidation. Metabolism of intramitochondrially generated branched chain alpha-keto acids was studied using [1-14C]leucine and [1-14C]valine. Essentially all of the labeled branched chain alpha-keto acid produced by transamination of [1-14C]leucine or [1-14C]valine with a low concentration of unlabeled branched chain alpha-keto acid (20 microM) was oxidized. Further addition of alpha-ketoglutarate resulted in a significant increase in the rate of labeled leucine or valine transamination, but again most of the labeled keto acid product was oxidized. Thus, catabolism of branched chain amino acids will be favored by a high concentration of mitochondrial alpha-ketoglutarate and low intramitochondrial glutamate.     

Effect of aromatic acids on protein synthesis in subcellular preparations from the rat brain.

The incorporation of [3H]phenylalanine, [3H]tyrosine, and [3H]tryptophan into protein and amino acyl-tRNA was studied in cell-free preparations from rat brain. Tyrosine and tryptophan inhibited the incorporation of phenylalanine into protein, and tyrosine inhibited the incorporation of phenylalanine and tryptophan into amino acyl-tRNAs. In most cases, homogentisate, phenylpyruvate, and phenyllactate inhibited the incorporation of phenylalanine, tyrosine, and tryptophan into protein and amino acyl-tRNAs, and the incorporation of phenylalanine into polyphenylalanine. All other protein amino acids, and phenylacetate, salicylate, and benzoate were wholly ineffectual. The results suggest that the formation of amino acyl-tRNAs may have been the step which was affected most by the inhibitors. The incorporation data at different concentrations of the aromatic amino acids were fitted to the simple Michaelis equation. Homogentisate and phenylpyruvate generally tended to reduce both Km and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than Km.     

Indole-3-Pyruvic Acid as an Intermediate in the Conversion of Tryptophan to Indole-3-Acetic Acid. I. Some Characteristics of Tryptophan Transaminase from Mung Bean Seedlings

Seedlings of mung bean (Phaseolus aureus) contain a soluble enzyme capable of converting l-tryptophan to indole-3-pyruvic acid by transamination. The concentration of the enzyme is highest in the stem meristem and primary leaves and lowest in the roots. The enzyme was purified 28.6 fold by ammonium sulphate precipitation, Sephadex G-200 filtration, and electrophoresis. The isoelectric point of the enzyme protein was pH 6.6. The optimum pH and temperature for the catalytic conversion were ca. 8.5 and 53°C respectively. Using l -tryptophan and α-ketoglutarate as substrates Km was found to be 3.3 × 10^?4 M and the activation energy 18,270 cal per mole. The enzyme converted only the l -form of tryptophan, phenylalanine, tyrosine, and histidine. Out of 13 other l -amino acids tested 8 could be transaminated. Eight α-keto acids tested could all be used as substrates. High efficiency of an α-keto acid as an amino group acceptor agreed usually with high efficiency of the corresponding amino acid as a donor. The pari ß-methyl-α-ketoisovaleric acid and isoleucine was an exception to that rule. Addition of pyridoxalphosphate to the reaction mixture was not needed. The indole-3-pyruvic acid formed in the reaction was trapped and partly stabilized as its borate complex and measured spectrophotometrically at 327 nm. The keto acid formed was further identified by chromatography of its 2,4-dinitrophenylhydrazone in 4 solvent systems. When using α-keto-glutaric acid as a substrate, the glutamic acid produced was determined by the glutamate dehydrogenase method. The sensitivity of the assay permits enzyme determinations in extracts from 5 mg leaves or 100 mg roots.     

Enzymatic transamination of D-kynurenine generates kynurenic acid in rat and human brain

In the mammalian brain, the α7 nicotinic and NMDA receptor antagonist kynurenic acid is synthesized by irreversible enzymatic transamination of the tryptophan metabolite l-kynurenine. d-kynurenine, too, serves as a bioprecursor of kynurenic acid in several organs including the brain, but the conversion is reportedly catalyzed through oxidative deamination by d-amino acid oxidase. Using brain and liver tissue homogenates from rats and humans, and conventional incubation conditions for kynurenine aminotransferases, we show here that kynurenic acid production from d-kynurenine, like the more efficient kynurenic acid synthesis from l-kynurenine, is blocked by the aminotransferase inhibitor amino-oxyacetic acid. In vivo, focal application of 100 μM d-kynurenine by reverse microdialysis led to a steady rise in extracellular kynurenic acid in the rat striatum, causing a 4-fold elevation after 2 h. Attesting to functional significance, this increase was accompanied by a 36% reduction in extracellular dopamine. Both of these effects were duplicated by perfusion of 2 μM l-kynurenine. Co-infusion of amino-oxyacetic acid (2 mM) significantly attenuated the in vivo effects of d-kynurenine and essentially eliminated the effects of l-kynurenine. Thus, enzymatic transamination accounts in part for kynurenic acid synthesis from d-kynurenine in the brain. These results are discussed with regard to implications for brain physiology and pathology.     

The biochemical basis for growth inhibition by L-phenylalanine in Neisseria gonorrhoeae

Clinical isolates of Neisseria gonorrhoeae are commonly subject to growth inhibition by phenylpyruvate or by L-phenylalanine. A blockade of tyrosine biosynthesis is indicated since inhibition is reversed by either L-tyrosine or 4-hydroxyphenylpyruvate. Phenylalanine-resistant (PheR) and phenylalanine-sensitive (PheS) isolates both have a single 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase that is partially inhibited by L-phenylalanine (80%). However, PheS and PheR isolates differ in that the ratio of phenylpyruvate aminotransferase to 4-hydroxyphenylpyruvate aminotransferase is distinctly greater in PheS isolates than in PheR isolates. A mechanism for growth inhibition is proposed in which phenylalanine exerts two interactive effects. (i) Phenylalanine decreases precursor flow to 4-hydroxyphenylpyruvate through its controlling effect upon DAHP synthase; and (ii) phenylalanine is largely transaminated to phenylpyruvate, which saturates both aminotransferases, preventing transamination of an already limited supply of 4-hydroxyphenylpyruvate to L-tyrosine.     

Effect of aromatic acids on protein synthesis in subcellular preparations from the rat brain

The incorporation of [³H]phenylalanine, [³H]tyrosine, and [³H]tryptophan into protein and amino acyl–tRNA was studied in cell-free preparations from rat brain. Tyrosine and tryptophan inhibited the incorporation of phenylalanine into protein, and tyrosine inhibited the incorporation of phenylalanine and tryptophan into amino acyl–tRNAs. In most cases, homogentisate, phenylpyruvate, and phenyllactate inhibited the incorporation of phenylalanine, tyrosine, and tryptophan into protein and amino acyl–tRNAs, and the incorporation of phenylalanine into polyphenylalanine. All other protein amino acids, and phenylacetate, salicylate, and benzoate were wholly ineffectual. The results suggest that the formation of amino acyl–tRNAs may have been the step which was affected most by the inhibitors. The incorporation data at different concentrations of the aromatic amino acids were fitted to the simple Michaelis equation. Homogentisate and phenylpyruvate generally tended to reduce both K_m and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than K_m.     

Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases.

Two new mutations are described which, together, eliminate essentially all the aminotransferase activity required for de novo biosynthesis of tyrosine, phenylalanine, and aspartic acid in a K-12 strain of Escherichia coli. One mutation, designated tyrB, lies at about 80 min on the E. coli map and inactivates the "tyrosine-repressible" tyrosine/phenylalanine aminotransferase. The second mutation, aspC, maps at about 20 min and inactivates a nonrespressible aspartate aminotransferase that also has activity on the aromatic amino acids. In ilvE- strains, which lack the branched-chain amino acid aminotransferase, the presence of either the tyrosine-repressible aminotransferase or the aspartate aminotransferase is sufficient for growth in the absence of exogenous tyrosine, phenylalanine, or aspartate; the tyrosine-repressible enzyme is also active in leucine biosynthesis. The ilvE gene product alone can reverse a phenylalanine requirement. Biochemical studies on extracts of strains carrying combinations of these aminotransferase mutations confirm the existence of two distinct enzymes with overlapping specificities for the alpha-keto acid analogues of tyrosine, phenylalanine, and aspartate. These enzymes can be distinguished by electrophoretic mobilities, by kinetic parameters using various substrates, and by a difference in tyrosine repressibility. In extracts of an ilvE- tyrB- aspC- triple mutant, no aminotransferase activity for the alpha-keto acids of tyrosine, phenylalanine, or aspartate could be detected.