Subcellular distribution and properties of kynurenine pyruvate transaminase in rat kidney.

Kynurenine transaminase activity in rat kidney was found in both the mitochondrial and supernatant fractions. These fractions contained (a) kynurenine pyruvate transaminase, which showed a preference for pyruvate as amino acceptor, and had a pH optimum between 8.0 and 8.5, and (b) kynurenine 2-oxoglutarate transaminase, with a preference for 2-oxoglutarate and a pH optimum between 6.0 and 6.5. The apparent Km value of the former enzyme for L-kynurenine was much lower than that of the latter enzyme.     

Peripheral distribution of kynurenine metabolites and activity of kynurenine pathway enzymes in renal failure.

We investigated L-kynurenine distribution and metabolism in rats with experimental chronic renal failure of various severity, induced by unilateral nephrectomy and partial removal of contralateral kidney cortex. In animals with renal insufficiency the plasma concentration and the content of L-tryptophan in homogenates of kidney, liver, lung, intestine and spleen were significantly decreased. These changes were accompanied by increase activity of liver tryptophan 2,3-dioxygenase, the rate-limiting enzyme of kynurenine pathway in rats, while indoleamine 2,3-dioxygenase activity was unchanged. Conversely, the plasma concentration and tissue content of L-kynurenine, 3-hydroxykynurenine, and anthranilic, kynurenic, xanthurenic and quinolinic acids in the kidney, liver, lung, intestine, spleen and muscles were increased. The accumulation of L-kynurenine and the products of its degradation was proportional to the severity of renal failure and correlated with the concentration of renal insufficiency marker, creatinine. Kynurenine aminotransferase, kynureninase and 3-hydroxyanthranilate-3,4-dioxygenase activity was diminished or unchanged, while the activity of kynurenine 3-hydroxylase was significantly increased. We conclude that chronic renal failure is associated with the accumulation of L-kynurenine metabolites, which may be involved in the pathogenesis of certain uremic syndromes.     

Measurement of Rat Brain Kynurenine Aminotransferase at Physiological Kynurenine Concentrations

The production of the neuroinhibitory and neuroprotective metabolite kynurenic acid (KYNA) was investigated in rat brain by examining its biosynthetic enzyme, kynurenine aminotransferase (KAT). By using physiological (low micromolar) concentrations of the substrate L-kynurenine (KYN) and by determining the irreversible conversion of [3H]KYN to [3H]KYNA as a measure of KAT activity, a novel, simple, and sensitive assay was developed which permitted the detailed characterization of the enzyme. Only a single protein, which under routine assay conditions showed approximately equal activity with 2-oxoglutarate and pyruvate as the aminoacceptor, was found in rat brain. The enzyme was distributed heterogeneously between the nine brain regions studied, with the KAT-rich olfactory bulb displaying approximately five times higher activity than the cerebellum, the area with lowest KAT activity. In subcellular fractionation studies, the majority of KAT was recovered in mitochondria. In contrast to many known aminotransferases, partially purified KAT was shown to be highly substrate-specific. Thus, of the amino acids tested, only alpha-aminoadipate and tryptophan displayed moderate competition with KYN. Notably, 3-hydroxykynurenine, reportedly a very good substrate of KAT, competed rather poorly with KYN as well. Aminooxyacetic acid, a nonspecific transaminase inhibitor, blocked KAT activity with an apparent Ki of 5 microM. Kinetic analyses with partially purified rat brain KAT revealed a Km of 17 microM for KYN with 1 mM 2-oxoglutarate, but a much higher Km (910 microM) with 1 mM pyruvate. Km values for 2-oxoglutarate and pyruvate were 150 and 160 microM, respectively. The cellular localization of KAT was examined in striatal homogenates obtained from rats 7 days after an intrastriatal injection of quinolinate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of kynurenine hydrolysis catalyzed by kynureninase.

Several kynurenine analogs have been prepared and examined for their susceptibility to hydrolytic cleavage by bacterial kynureninase. In addition to L-kynurenine, 4-fluoro- and 5-fluoro-L-kynurenines were hydrolyzed rapidly. 3-Hydroxy-, 5-hydroxy-, 5-methyl-, and N'-formyl-L-kynurenines, and beta-benzoyl-DL-alanine were hydrolyzed slowly, whereas D-kynurenine, S-benzyl-L-cysteine, and L-asparagine were not hydrolyzed. Kinetic parameters for these kynurenine analogs indicate that a substituent on the benzene ring of kynurenine does not greatly affect the affinity of the enzyme for the substrate but does markedly affect the rate of hydrolysis. gamma-(o-Aminophenyl)-L-homoserine was converted into L-alanine and o-amino-benzaldehyde, suggesting that the sigma-bond electrons between the beta- and gamma-carbon atoms of this kynurenine analog remain in the alanyl moiety during the enzyme reaction. Aromatic compounds such as o-aminobenzaldehyde and o-aminoacetophenone strongly inhibited the kynurenine hydrolysis. It was shown that kynurenic acid is not produced by kynureninase by the use of isotopically labeled substrate. A small amount of pyruvate was definitely formed in the kynureninase reaction. On the basis of these results, a reaction mechanism is proposed for the enzymatic kynurenine cleavage, involving hydrolysis of the alpha, gamma-diketone intermediate to give anthranilic acid and the pyruvate-pyridoxamine 5'-phosphate Schiff base, which is further converted into the alanine-pyridoxal 5'-phosphate Schiff base, or directly hydrolyzed to give pyruvate and the pyridoxamine 5'-phosphate form of the enzyme.     

Participation of kynurenine and its derivatives in disorders of the cardiac rhythm

The influence of tryptophan derivatives: L-kynurenine, 3-hydroxy-DL-kynurenine, kynurenic, xanthurenic, 3-hydroxy-anthranilic and quinolinic acids has been investigated in isolated frog heart. It has been established that L-kynurenine, 3-hydroxy-DL-kynurenine, xanthurenic acid and quinolinic acid at a concentration of 10(-6)-10(-3) mol/l initiate bradycardia. In some cases xanthurenic and quinolinic acids cause a one-minute cardiac arrest in early diastole. 3-hydroxy-anthranilic acid at a concentration of 10(-5) and 5 X 10(-5) mol/l produces premature beats and attacks of tachycardia. In the experiments, using 10(-6)-10(-3) mol/l of kynurenic acid, no impairment of the cardiac rhythm was observed in the isolated frog heart.     

Purification and characterization of kynurenine--2-oxoglutarate aminotransferase from the liver, brain and small intestine of rats.

1. Kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) was purified to homogeneity from the liver, brain and small intestine of rats by the same procedure. The three enzyme preparations had nearly identical pH optima, substrate specificities and molecular weights. Isoenzyme 1 was active with 2-oxoglutarate but not with pyruvate as amino acceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order to activity: L-aspartate greater than L-tyrosine greater than L-phenylalanine greater than L-tryptophan greater than 5-hydroxy-L-tryptophan greater than L-kynurenine. The molecular weight was approximately 88 000 as determined by sucrose-density-gradient centrifugation. The pH optimum was between 8.0 and 8.5. On the basis of substrate specificity, substrate inhibition, subcellular distribution and polyacrylamide-disc-gel electrophoresis, it is suggested that liver, brain and small intestinal kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) is identical with mitochondrial tyrosine-2-oxoglutarate aminotransferase and also with mitochondrial aspartate-2-oxoglutarate aminotransferase. 2. An additional kynurenine-2-oxoglutarate aminotransferase (isoenzyme 2) was purified from the liver. This enzyme was specific for 2-oxoglutarate and L-kynurenine. Sucrose-density-gradient centrifugation gave a molecular weight of approximately 100 000. The pH optimum was between 6.0 and 6.5. This enzyme was not detected in the brain or small intestine.     

Enzyme activities involved in tryptophan metabolism along the kynurenine pathway in rabbits

The following enzyme activities of the tryptophan-nicotinic acid pathway were studied in male New Zealand rabbits: liver tryptophan 2,3-dioxygenase, intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4-dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase. Intestine superoxide dismutase and serum tryptophan were also determined. Liver tryptophan 2,3-dioxygenase exists only as holoenzyme, but intestine indole 2,3-dioxygenase is very active and can be considered the key enzyme which determines how much tryptophan enters the kynurenine pathway also under physiological conditions. The elevated activity of indole 2,3-dioxygenase in the rabbit intestine could be related to the low activity of superoxide dismutase found in intestine. Kynurenine 3-monooxygenase appeared more active than kynurenine-oxoglutarate transaminase and kynureninase, suggesting that perhaps a major portion of kynurenine available from tryptophan may be metabolized to give 3-hydroxyanthranilic acid, the precursor of nicotinic acid. In fact, 3-hydroxyanthranilate 3,4-dioxygenase is much more active than the other previous enzymes of the kynurenine pathway. In the rabbit liver 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase show similar activities, but in the kidney 3-hydroxyanthranilate 3,4-dioxygenase activity is almost double. These data suggest that in rabbit tryptophan is mainly metabolized along the kynurenine pathway. Therefore, the rabbit can also be a suitable model for studying tryptophan metabolism in pathological conditions.     

Studies on the kynurenine adminotransferase activity in rat liver and kidney.

The kynurenine aminotransferase activity of supernatant and mitochondrial fractions obtained from rat liver and kidney was studied with L-kynurenine and L-3-hydroxykynurenine as substrates. A substrate inhibition with L-kynurenine at concentrations higher than 6-7mM was observed with all four enzyme preparations. This did not happen with L-3-hydroxykynurenine as a substrate. Moreover, the liver mitochondrial enzyme shows a Km for pyridoxal phosphate 2-4 times smaller than the other preparations when assayed with L-3-hydroxykynurenine as a substrate. Therefore, the accumulation of xanthurenic acid and not of kynurenic acid in B6 deficiency could be related both to this high activity of liver mitochondrial kynurenine aminotransferase with L-3-hydroxykynurenine, even at small concentrations of B6, and to substrate inhibition observed with L-kynurenine and not with L-3-hydroxykynurenine.     

2-Oxoacids Regulate Kynurenic Acid Production in the Rat Brain

Abstract : This study was designed to examine the role of 2-oxoacids in the enzymatic transamination of L-kynurenine to the excitatory amino acid receptor antagonist, kynurenate, in the rat brain. In brain tissue slices incubated in Krebs-Ringer buffer with a physiological concentration of L-kynurenine, pyruvate, and several other straight- and branched-chain 2-oxoacids, substantially restored basal kynurenate production in a dose-dependent manner without increasing the intracellular concentration of L-kynurenine. All 2-oxoacids tested also reversed or attenuated the hypoglycemia-induced decrease in kynurenate synthesis, but only pyruvate and oxaloacetate also substantially restored intracellular L-kynurenine accumulation. Thus, 2-oxoacids increase kynurenate formation in the brain primarily by functioning as co-substrates of the transamination reaction. This was supported further by the fact that the nonspecific kynurenine aminotransferase inhibitors (aminooxy)acetic acid and dichlorovinylcysteine prevented the effect of pyruvate on kynurenate production in a dose-dependent manner. Moreover, all 2-oxoacids tested attenuated or prevented the effects of veratridine, quisqualate, or L-α-aminoadipate, which reduce the transamination of L-kynurenine to kynurenate. Finally, dose-dependent increases in extracellular kynurenate levels in response to an intracerebral perfusion with pyruvate or α-ketoisocaproate were demonstrated by in vivo microdialysis. Taken together, these data show that 2-oxoacids can directly augment the de novo production of kynurenate in several areas of the rat brain. 2-Oxoacids may therefore provide a novel pharmacological approach for the manipulation of excitatory amino acid receptor function and dysfunction.     

Identity of kynurenine: pyruvate aminotransferase with histidine: pyruvate aminotransferase.

Kynurenine pyruvate aminotransferase was purified from rat kidney. The purified enzyme had an isoelectric point of pH 5.2 and a pH optimum of 9.3. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors. L-Amino acids were effective in the following order of activity: histidine greather than phenylalanine greater than kynurenine greater than tyrosine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values were about 0.63 mM, 1.4 mM and 0.09 mM for histidine, kynurenine and phenylalanine, respectively. Km values for pyruvate were 5.5 mM with histidine as amino donor, 1.3 mM with kynurenine and 8.5 mM with phenylalanine. Kynurenine pyruvate aminotransferase activity of the enzyme was inhibited by the addition of histidine or phenylalanine. The molecular weights determined by gel filtration and sucrose density gradient centrifugation were approximately 76000 and 79000, respectively. On the basis of purification ratio, substrate specificity, inhibition by common substrates, subcellular distribution, isoelectric focusing and polyacrylamide-gel electrophoresis, it is suggested that kynurenine pyruvate aminotransferase is identical with histidine pyruvate aminotransferase and also with phenylalanine pyruvate aminotransferase. The physiological significance of the enzyme is discussed.     

Preparation and photophysical properties of a caged kynurenine

We have prepared l-kyurenine 4-hydroxyphenacyl ester, a caged derivative of L-kynurenine. N(α)-tBOC-L-tryptophan was reacted with 4-hydroxyphenacyl bromide in DMF with K(2)CO(3) as the base to give the N(α)-tBOC 4-hydroxyphenacyl ester. The ester was then treated with O(3) in MeOH at -20°C, followed by trifluoroacetic acid in CH(2)Cl(2), then aqueous HCl to obtain the caged kynurenine as the dihydrochloride salt. The caged kynurenine is stable as a dry solid in the dark at -78°C, but in aqueous solutions in phosphate buffer at pH 7-8 hydrolyzes rapidly (t(1/2) ～5 min). Solutions in Tris at pH 7 are more stable (t(1/2) >30 min), and solutions in 1mM HCl are stable for several hours. As expected, the ester is cleaved in microseconds with laser pulses at 355 nm. The caged kynurenine may be useful for preparation of substrate complexes for crystallography or in biological studies on kynurenine.     

Cloricromene effect on the enzyme activities of the tryptophan-nicotinic acid pathway in diabetic/hyperlipidemic rabbits

Since alterations of tryptophan metabolism have been reported in diabetes and atherosclerosis, it was thought of interest to investigate any role of cloricromene through the influence on the oxidative metabolism of the amino acid by using diabetic/hyperlipidemic rabbits.Male 4-month-old New Zealand white rabbits, fed a diet enriched with 1% cholesterol and 10% corn oil, were made diabetic with alloxan. During the hyperlipidemic diet, a group of rabbits was treated with cloricromene (10 mg/kg/day subcutaneously plus 1.5 mg/kg/day intravenously, for 5 weeks). The other group received saline. Normometabolic New Zealand rabbits fed standard diet, treated or not with cloricromene, were used as control.The specific activities of liver tryptophan 2,3-dioxygenase and small intestine indole 2,3-dioxygenase were not significantly changed by the drug treatment. Also the specific activities of other enzymes of the kynurenine pathway in the liver and kidneys, specifically kynurenine 3-monooxygenase, kynureninase and kynurenine-oxoglutarate transaminase, did not show any significant difference in both tissues between the two groups of rabbits. On the contrary, 3-hydroxyanthranilate 3,4-dioxygenase activity in the liver of diabetic/hyperlipidemic rabbits and control rabbits treated with cloricromene showed a slight increase in comparison with untreated animals. Conversely, the specific activity of the enzyme in kidneys was not affected by the drug treatment in diabetic/hyperlipidemic animals but was reduced in controls. Aminocarboxymuconate-semialdehyde decarboxylase specific activity remained unchanged in the liver following cloricromene treatment, instead the specific activity of the enzyme in the kidneys of the diabetic/hyperlipidemic rabbits was significantly increased by the drug, with a value more than double in comparison to untreated animals. The activity of the scavenger enzyme Cu/Zn superoxide dismutase (Cu/Zn SOD) in the small intestine was also determined and found significantly increased of about twice as much in the group of diabetic/hyperlipidemic rabbits treated with cloricromene.In conclusion, in diabetic/hyperlipidemic rabbits, cloricromene appeared to influence the enzymes involved in the last steps of tryptophan oxidative metabolism through the kynurenine pathway. This, together with the antioxidant action through the activation of Cu/Zn SOD, might deserve further investigation for evaluating any link between the observed experimental findings at the level of the kynurenine pathway and the clinical effect of the drug.     

A radiometric kynurenine monooxygenase assay

Kynurenine 3-monooxygenase is a flavin-dependent monooxygenase that catalyzes the oxidation of L-kynurenine to 3-hydroxy-L-kynurenine in the kynurenine pathway of tryptophan metabolism. The enzyme requires NADH or NADPH as a cofactor. A discontinuous assay that utilizes L-[3H]kynurenine as substrate is described. The assay offers high precision and a wide range of accessible substrate and cofactor concentrations. The assay was used to measure kinetic isotope effects and the stereospecificity of oxidation of the cofactor. Hydride is transferred from the A-side (pro-R) of NADH and NADPH since primary deuterium isotope effects were observed for both cofactors when they were deuterated on the A-side but not on the B-side. The large isotope effect on Vmax/Km for NADH is sensitive to the concentration of kynurenine, which indicates that NADH can bind before kynurenine.     

Presence of Kynurenine Hydroxylase in Developing Rat Brain

Abstract: Kynurenine-3-hydroxylase, an enzyme that is part of the degradative pathway for tryptophan, was present in the cerebral cortex of neonatal rats and exhibited a K_m , for L-kynurenine close to that of the liver enzyme. This enzyme was enriched in mitochondrial fractions isolated from cerebral cortices of neonatal rats by Ficoll-sucrose gradient centrifugation, with some activity also present in synaptosomal fractions probably due to the mitochondrial content of synaptosomes since cytochrome c oxidase, another mitochondrial enzyme, had a similar distribution in the gradient. Kynurenine hydroxylase as well as monoamine oxidase, another mitochondrial enzyme, had increased specific activities in synaptosomal fractions isolated from 14-day-old rats compared to fractions from 8-day-old rats. Hypothyroidism, induced on the day of birth, resulted in increased activities of kynurenine hydroxylase and monoamine oxidase in synaptosomal fractions isolated from 14-day-old rats.     

Analysis of kynurenine transaminase activity in Drosophila by high performance liquid chromatography

A sensitive assay for kynurenine transaminase activity (E.C. 2.6.1.7) based on rapid separation of the reaction product by high performance liquid chromatography (HPLC) has been developed. Drosophila sordidula extracts have been assayed by this new method and this is the first time that kynurenine transaminase activity has been demonstrated in Drosophila. The method of assay developed can be extended to any other organism. Kynurenine and 3-hydroxykynurenine were both used as substrates, and they were transaminated to kynurenic acid and xanthruenic acid, respectively. HPLC is used to separate and quantitate these reaction products from all other components in the reaction mixture.In crude extracts from Drosophila, the reaction requires pyridoxal 5′-phosphate and an amino acid acceptor. The enzyme activity showed a maximum at 47°C and pH 8.0 with kynurenine and pyruvic acid as substrates. Transaminase activity was present in both head and body, nevertheless the specific activity was higher in the former. In bodies, pyruvic acid was the best amino acceptor whereas in heads it was α-oxoglutaric acid. The variation of kynurenine transaminase during development of D. sordidula showed, in the larval and pupal stages, activity levels practically constant and much lower than those found in the adult. This seems to suggest a preferential role of this enzyme in the metabolism of intermediates in the biosynthesis of ommochromes.     

Rat Brain Slices Produce and Liberate Kynurenic Acid upon Exposure to l-Kynurenine

The incorporation of L-kynurenine (L-KYN) into kynurenic acid (KYNA) was examined in rat brain slices. KYNA was measured in the slices and in the incubation medium after purification by ion-exchange and HPLC chromatography. In pilot experiments, the formation of KYNA was confirmed by gas chromatography. KYNA was produced stereoselectively from L-KYN, and approximately 90% of the newly synthesized KYNA was recovered from the incubation medium. Intracellular KYNA was not actively retained by the tissue and was lost from the cells upon repeated washes. Thus, regulation of the levels of extracellular KYNA appears to occur at the level of L-KYN uptake and/or kynurenine transaminase, the biosynthetic enzyme of KYNA. KYNA production from L-KYN was linear up to 4 h and reached a plateau at a L-KYN concentration of 250 microM. The process was effectively inhibited by the transaminase inhibitor aminooxyacetic acid (IC50, approximately 25 microM), and showed pronounced regional distribution (hippocampus greater than cortical areas greater than thalamus much greater than cerebellum). The conversion of L-KYN to KYNA was dependent on oxygenation and on the presence of glucose in the incubation medium. Neither deletion of Ca2+ or Mg2+ nor addition of 20 mM Mg2+ had any effect. However, KYNA production was significantly attenuated in the absence of Cl- or in the presence of 50 mM K+ in the incubation medium. In Na+-free medium, the production of KYNA from L-KYN was increased by 30%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of glutamate on the control of fatty-acid synthesis in white adipose tissue of the rat. Inhibition of pyruvate dehydrogenase.

Glutamate (5mM) inhibited glucose conversion to fatty acids by approximately one-third in adipocytes from fed rats. This inhibition was significantly less in the pressence of pyruvate or 2-oxoglutarate. After incubation of adipose tissue from fed rats with glucose and insulin, pyruvate dehydrogenase activity was 180 plus or minus 17 mU/g wet weight. Addition of glutamine to the incubation medium decreased this activity significantly (118 plus or minus 14 mU/g wet weight). This inhibition by glutamate was also diminished when 2-oxoglutarate or pyruvate were present. Glutamate added to homohentates of adipose tissue had no effect on the activation of pyruvate dehydrogenase by Mg-2+. However, glutamate inhibited the active form of the enzyme and enhanced the rate of inactivation of the enzyme complex by ATP and Mg-2+. Aminooxyacetate, a transaminase inhibitor, did not reverse the effects of glutamate on pyruvate dehydrogenase nor fatty acid synthesis.     

Enzyme activities along the tryptophan-nicotinic acid pathway in alloxan diabetic rabbits

Recent data from our laboratory have indicated that the rabbit is a suitable animal model for the study of enzyme activities of the tryptophan-nicotinic acid pathway. We report here the pattern of tryptophan metabolism in rabbits made diabetic with alloxan treatment, and hypercholesterolemic with a high-cholesterol diet. A group of rabbits with only hypercholesterolemia was also considered. The enzymes assayed were: liver tryptophan 2,3-dioxygenase (TDO), intestine indoleamine 2,3-dioxygenase (IDO), liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase.TDO showed a reduction of specific activity in liver of diabetic-hyperlipidemic and hyperlipidemic rabbits compared to controls. Intestine IDO activities and liver and kidney kynurenine monooxygenase were unchanged with respect to controls.Kynurenine-oxoglutarate transaminase and kynureninase activities were reduced in the kidneys, but not in the liver, of diabetic-hyperlipidemic rabbits.The main finding was the reduction of 3-hydroxyanthranilate 3,4-dioxygenase activity (expressed as activity per g of fresh tissue) in the liver and kidneys of diabetic-hypercholesterolemic and hyperlipidemic rabbits compared to controls. Conversely, aminocarboxymuconate-semialdehyde decarboxylase activity was significantly higher in diabetic hypercholesterolemic rabbits in comparison with control and hypercholesterolemic rabbits.These data demonstrate that also in diabetic rabbits there is an alteration of tryptophan metabolism at the level of 3-hydroxyanthranilic acid-->nicotinic acid step. Also dyslipidemia seems to be involved in enzyme activity variations of the tryptophan metabolism along the kynurenine pathway.     

Pyruvate decarboxylating action of L-cycloserine. The significance of this in understanding its metabolic inhibitory action

We present evidence which demonstrates that L-cycloserine, structural analog of L-alanine, which is known to be an effective aminotransferase inhibitor, is also a potent inhibitor of cellular pyruvate metabolism. This effect was found to be related to its almost instantaneous action in decreasing pyruvate concentrations in a dose-dependent manner. 1H nuclear magnetic resonance studies clearly demonstrate that the irreversible removal of pyruvate induced by L-cycloserine is caused by the decarboxylating action of the latter. Pyruvate disappearance induced by L-cycloserine can be stoichiometrically accounted for as acetate. The process does not involve any chemically detected transformation of L-cycloserine. These observations lead to two main considerations regarding the known action of L-cycloserine. First, its inhibitory effect on gluconeogenesis from lactate could be explained only on the basis of its ability to reduce pyruvate availability with no apparent need for transaminase inhibition. Second, its ability as a transaminase inhibitor should be reconsidered in view of its potent decarboxylating action on pyruvate and probably other oxoacids.     

pH dependence, substrate specificity and inhibition of human kynurenine aminotransferase I.

Human kynurenine aminotransferase I/glutamine transaminase K (hKAT-I) is an important multifunctional enzyme. This study systematically studies the substrates of hKAT-I and reassesses the effects of pH, Tris, amino acids and alpha-keto acids on the activity of the enzyme. The experiments were comprised of functional expression of the hKAT-I in an insect cell/baculovirus expression system, purification of its recombinant protein, and functional characterization of the purified enzyme. This study demonstrates that hKAT-I can catalyze kynurenine to kynurenic acid under physiological pH conditions, indicates indo-3-pyruvate and cysteine as efficient inhibitors for hKAT-I, and also provides biochemical information about the substrate specificity and cosubstrate inhibition of the enzyme. hKAT-I is inhibited by Tris under physiological pH conditions, which explains why it has been concluded that the enzyme could not efficiently catalyze kynurenine transamination. Our findings provide a biochemical basis towards understanding the overall physiological role of hKAT-I in vivo and insight into controlling the levels of endogenous kynurenic acid through modulation of the enzyme in the human brain.