The protease problem in Neurospora. Variable stability of enzymes in aromatic amino acid metabolism.

A proteolytic activity isolated from Neurospora crassa is shown to be responsible for the variable stability observed in vitro for enzymes involved in aromatic amino acid metabolism. For example, the activity of kynurenine formamidase was insensitive to the action of this protease preparation over a 24-h period of incubation at 25 °C, whereas chorismate synthase, anthranilate synthase, kynureninase, and the five activities of the arom multienzyme system were inactivated during this time. Anthranilate synthase and two of the arom system activities (dehydroquinate synthase and shikimate kinase) were inactivated by the protease preparation within 2 h. Phenylmethanesulfonylfluoride and a specific proteolytic inhibitor from N. crassa prevented inactivation of these enzymes. Spontaneous loss of activity at 25 °C of purified samples of anthranilate synthase, dehydroquinate synthase and shikimate kinase was also prevented by the inhibitors. A method for purifying the inhibitor from N. crassa is described, and its use as a reagent in the analysis of proteolytic action is demonstrated.     

Early steps of metabolism evolution inferred by cladistic analysis of amino acid catabolic pathways

Among abiotic molecules available in primitive environments, free amino acids are good candidates as the first source of energy and molecules for early protocells. Amino acid catabolic pathways are likely to be one of the very first metabolic pathways of life. Among them, which ones were the first to emerge? A cladistic analysis of catabolic pathways of the sixteen aliphatic amino acids and two portions of the Krebs cycle is performed using four criteria of homology. The cladogram shows that the earliest pathways to emerge are not portions of the Krebs cycle but catabolisms of aspartate, asparagine, glutamate, glutamine, proline, arginine. Earliest enzymatic catabolic functions were deaminations and transaminations. Later on appeared enzymatic decarboxylations. The consensus tree allows to propose four time spans for catabolism development and corroborates the views of Cordón in 1990 about the evolution of catabolism.     

Gender differences in the regulation of amino acid metabolism.

Exercising men, compared with women, have a greater increase in leucine oxidation but not lysine rate of appearance. The cause for this sexual dimorphism is unknown; however, an inhibition of beta-adrenoreceptor activity has previously been shown to mediate amino acid metabolism (Lamont LS, McCullough AJ, and Kalhan SC. Am J Physiol Endocrinol Metab 268: E910-E916, 1995; Lamont LS, Patel DG, and Kalhan SC. J Appl Physiol 67: 221-225, 1989). This study was a gender comparison of leucine and lysine kinetics during a beta-adrenoreceptor blockade (beta1,beta2-blockade) and a placebo control by using a double-blind crossover protocol. Subjects exercised at 50% of their trial-specific maximal O2 consumption (1 h) after 7 days of dietary control. During exercise with beta-blockade, men had an increased nonprotein respiratory exchange ratio (P < 0.001), whereas women had an increased circulation of free fatty acids (P < 0.001). The genders also displayed distinct differences in exercise amino acid kinetics. The men, but not the women, increased leucine oxidation (P < 0.005) and lysine rate of appearance (P < 0.009) when exercising during beta-adrenergic blockade. This study indicates that during beta-blockade, exercising men increase their need for amino acids (and carbohydrate) to fuel energy needs, whereas women increase their mobilization of fat, thereby requiring less alternative fuels such as carbohydrate and amino acids. Gender-specific fuel preferences during exercise are regulated by beta-adrenergic-receptor activity. Substrate availability during exercise appears to modulate the amino acid oxidation differences between genders.     

Short-term fasting, seizure control and brain amino acid metabolism

The ketogenic diet is an effective treatment for seizures, but the mechanism of action is unknown. It is uncertain whether the anti-epileptic effect presupposes ketosis, or whether the restriction of calories and/or carbohydrate might be sufficient. We found that a relatively brief (24 h) period of low glucose and low calorie intake significantly attenuated the severity of seizures in young Sprague-Dawley rats (50-70 gms) in whom convulsions were induced by administration of pentylenetetrazole (PTZ). The blood glucose concentration was lower in animals that received less dietary glucose, but the brain glucose level did not differ from control blood [3-OH-butyrate] tended to be higher in blood, but not in brain, of animals on a low-glucose intake. The concentration in brain of glutamine increased and that of alanine declined significantly with low-glucose intake. The blood alanine level fell more than that of brain alanine, resulting in a marked increase ( approximately 50%) in the brain:blood ratio for alanine. In contrast, the brain:blood ratio for leucine declined by about 35% in the low-glucose group. When animals received [1-(13)C]glucose, a metabolic precursor of alanine, the appearance of (13)C in alanine and glutamine increased significantly relative to control. The brain:blood ratio for [(13)C]alanine exceeded 1, indicating that the alanine must have been formed in brain and not transported from blood. The elevated brain(alanine):blood(alanine) could mean that a component of the anti-epileptic effect of low carbohydrate intake is release of alanine from brain-to-blood, in the process abetting the disposal of glutamate, excess levels of which in the synaptic cleft would contribute to the development of seizures.     

Selective enrichment of aromatic amino acid auxotrophs in Hansenula polymorpha.

Aromatic amino acid auxotrophs of the methanol-utilizing yeast Hansenula polymorpha were effectively selected by the use of nystatin and a medium that inhibits the growth of tyrosine auxotrophs. The procedure resulted in a frequency of aromatic auxotrophs of 2% of survivors and an enrichment of 20-fold. The new procedure also takes less time than traditional procedures. Of the auxotrophic mutants isolated, two-thirds required tyrosine and the remainder were tyrosine-phenylalanine double auxotrophs.     

Selective enrichment of aromatic amino acid auxotrophs in Hansenula polymorpha.

Aromatic amino acid auxotrophs of the methanol-utilizing yeast Hansenula polymorpha were effectively selected by the use of nystatin and a medium that inhibits the growth of tyrosine auxotrophs. The procedure resulted in a frequency of aromatic auxotrophs of 2% of survivors and an enrichment of 20-fold. The new procedure also takes less time than traditional procedures. Of the auxotrophic mutants isolated, two-thirds required tyrosine and the remainder were tyrosine-phenylalanine double auxotrophs.     

The metabolism of L-tryptophan by isolated rat liver cells. Quantification of the relative importance of, and the effect of nutritional status on, the individual pathways of tryptophan metabolism.

1. The metabolism of L-tryptophan by liver cells prepared from fed and 48 h-starved rats was studied. Methods are described, with the use of L-[ring-2-(14)C], L-[carboxy-14C]-and L-[benzene-ring-U-14C]-tryptophan, for the simultaneous determination of tryptophan 2,3-dioxygenase and kynureninase activities and of the oxidation of tryptophan to CO2 and non-aromatic intermediates of the kynurenine-glutarate pathway. 2. At physiological concentrations (0.1 mM), tryptophan was oxidized by tryptophan 2,3-dioxygenase at comparable rates in liver cells from both fed and starved rats. Kynureninase activity of hepatocytes from starved rats was 50% greater than that of cells from fed rats. About 10% of the tryptophan metabolized by tryptophan 2,3-dioxygenase was degraded completely to CO2. 3. In the presence of 0.5 mM-L-tryptophan, tryptophan 2,3-dioxygenase and kynureninase activities increased 5--6-fold. Liver cells from starved rats oxidized tryptophan at about twice the rate of these from fed rats. Degradation of tryptophan to non-aromatic intermediates of the glutarate pathway and CO2 was increased only 3-fold, suggesting an accumulation of aromatic intermediates of the kynurenine pathway. 4. Rates of metabolism with 2.5 mM-L-tryptophan were not significantly different from those obtained with 0.5 mM-tryptophan. 5. Rates of synthesis of quinolinic acid from 0.5 mM-L-tryptophan, determined either by direct quantification or indirectly from rates of radioisotope release from L-[carboxy-(14)C]- and [benzene-ring-U-14C]tryptophan, were essentially similar. 6. At all three concentrations examined, tryptophan was degraded exclusively through kynurenine; there was no evidence of formation of either indol-3-ylacetic acid or 5-hydroxyindol-3-ylacetic acid.     

Regulation of aromatic amino acid biosynthesis in Escherichia coli K-12: control of the aroF-tyrA operon in the absence of repression control.

Evidence was found which indicated that a mutation in gene trpS affected the rate of synthesis of tyrosine-repressible 3-deoxy-D-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase. The effect was found to occur independently of repression mediated by the tyrR gene product, and it was not due to a change in growth rate, nor was it a manifestation of the stringent response. It is proposed that in the proximal region of the aroF-tyrA operon there is an attenuator site controlled by the level of charged tryptophanyl-transfer RNA. In addition, it was demonstrated that starvation for certain amino acids led to degradation of tyrosine-repressible DAHP synthetase, but not phenylalanine-repressible DAHP synthetase, and supplementation with the missing amino acid led to an increased rate of synthesis of tyrosine-repressible DAHP synthetase during subsequent growth.     

Molecular analysis of the role of two aromatic aminotransferases and a broad-specificity aspartate aminotransferase in the aromatic amino acid metabolism of Pyrococcus furiosus

The genes encoding aromatic aminotransferase II (AroAT II) and aspartate aminotransferase (AspAT) from Pyrococcus furiosus have been identified, expressed in Escherichia coli and the recombinant proteins characterized. The AroAT II enzyme was specific for the transamination reaction of the aromatic amino acids, and uses a-ketoglutarate as the amino acceptor. Like the previously characterized AroAT I, AroAT II has highest efficiency for phenylalanine (k(cat)/Km = 923 s(-1) mM(-1)). Northern blot analyses revealed that AroAT I was mainly expressed when tryptone was the primary carbon and energy source. Although the expression was significantly lower, a similar trend was observed for AroAT II. These observations suggest that both AroATs are involved in amino acid degradation. Although AspAT exhibited highest activity with aspartate and alpha-ketoglutarate (k(cat) approximately 105 s(-1)), it also showed significant activity with alanine, glutamate and the aromatic amino acids. With aspartate as the amino donor, AspAT catalyzed the amination of alpha-ketoglutarate, pyruvate and phenyl-pyruvate. No activity was detected with either branched-chain amino acids or alpha-keto acids. The AspAT gene (aspC) was expressed as a polycistronic message as part of the aro operon, with expression observed only when the aromatic amino acids were absent from the growth medium, indicating a role in the biosynthesis of the aromatic amino acids.