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.     

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.     

Functional characterization and mechanism of action of recombinant human kynurenine 3-hydroxylase.

The mitochondrial outer membrane enzyme kynurenine 3-hydroxylase (K3H) is an NADPH-dependent flavin mono-oxygenase involved in the tryptophan pathway, where it catalyzes the hydroxylation of kynurenine. K3H was transiently expressed in COS-1 cells as a glutathione S-transferase (GST) fusion protein, and the pure recombinant protein (rec-K3H) was obtained with a specific activity of about 2000 nmol.min-1.mg-1. Rec-K3H was shown to have an optimum pH at 7.5, to use NADPH more efficiently than NADH, and to contain one molecule of non-covalently bound FAD per molecule of enzyme. The mechanism of the rec-K3H-catalyzed reaction was investigated by overall initial-rate measurements, and a random mechanism in which combination of the enzyme with one substrate does not influence its affinity for the other is proposed. Further kinetic studies revealed that K3H activity was inhibited by both pyridoxal phosphate and Cl-, and that NADPH-catalyzed oxidation occurred even in the absence of kynurenine if 3-hydroxykynurenine was present, suggesting an uncoupling effect of 3-hydroxykynurenine with peroxide formation. This observation could be of clinical interest, as peroxide formation could explain the neurotoxicity of 3-hydroxykynurenine in vivo.     

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.     

Tyrosine hydroxylase assay for detection of low levels of enzyme activity in peripheral tissues

A nonisotopic assay for tyrosine hydroxylase, with optimized signal-to-noise ratios, enables determination of low levels of enzyme activity in peripheral tissues. DOPA produced by the enzyme is measured using HPLC with electrochemical detection. Increased signal-to-noise ratios are obtained by including in the reaction mixture glycerol for reduction of blank values and dihydropteridine reductase and NADPH for regeneration of the tetrahydropteridine cofactor. With this method, tyrosine hydroxylase activity can be detected in as few as 200 PC12 cells and in peripheral tissues at levels as low as 4.5 fmol/min/mg wet weight. The assay permits activity to be assessed in a variety of peripheral tissues.     

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.     

Identification of formyl kynurenine formamidase and kynurenine aminotransferase from Saccharomyces cerevisiae using crystallographic, bioinformatic and biochemical evidence

The essential enzymatic cofactor NAD+ can be synthesized in many eukaryotes, including Saccharomyces cerevisiae and mammals, using tryptophan as a starting material. Metabolites along the pathway or on branches have important biological functions. For example, kynurenic acid can act as an NMDA antagonist, thereby functioning as a neuroprotectant in a wide range of pathological states. N-Formyl kynurenine formamidase (FKF) catalyzes the second step of the NAD+ biosynthetic pathway by hydrolyzing N-formyl kynurenine to produce kynurenine and formate. The S. cerevisiae FKF had been reported to be a pyridoxal phosphate-dependent enzyme encoded by BNA3. We used combined crystallographic, bioinformatic and biochemical methods to demonstrate that Bna3p is not an FKF but rather is most likely the yeast kynurenine aminotransferase, which converts kynurenine to kynurenic acid. Additionally, we identify YDR428C, a yeast ORF coding for an alpha/beta hydrolase with no previously assigned function, as the FKF. We predicted its function based on our interpretation of prior structural genomics results and on its sequence homology to known FKFs. Biochemical, bioinformatics, genetic and in vivo metabolite data derived from LC-MS demonstrate that YDR428C, which we have designated BNA7, is the yeast FKF.     

Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity

These studies report the development of a simple, specific, and highly sensitive fluorometric assay for rat liver peroxisomal fatty acyl-CoA oxidase activity. In this in vitro procedure fatty acyl-CoA-dependent H2O2 production was coupled in a peroxidase-catalyzed reaction to the oxidation of scopoletin (6-methoxy-7-hydroxycoumarin), a highly fluorescent compound, to a nonfluorescent product. Enzyme-catalyzed reaction rates as low as 5 pmol of H2O2 produced per minute could readily be detected. The reaction was studied in liver homogenates from normal rats with respect to absolute activity, time course, protein concentration dependence, substrate concentration dependence, pH optimum, substrate specificity, and cofactor requirements. The properties of the enzyme activity as assessed by the fluorometric assay agree well with those determined by other investigators using other assay methods. After subcellular fractionation of liver homogenates by differential centrifugation, the fatty acyl-CoA oxidase activity distributed like known peroxisomal marker enzymes. These results demonstrate that the fluorometric assay of fatty acyl-CoA oxidase should be useful in studying the distribution, properties, and subcellular localization of the enzyme, particularly in enzyme sources of low activity or in situations when only small amounts of material are available.     

Determination of serum kynurenine and hepatic tryptophan dioxygenase activity by high-performance liquid chromatography

The status of the oxidative metabolism of L-tryptophan is usually evaluated by the determination of tryptophan metabolites in serum or urine and/or the activities of various oxidative enzymes in tissues. I have developed assays for serum kynurenine and hepatic tryptophan dioxygenase (TDO) activity based on the determination of kynurenine (KYN) by isocratic, reverse phase HPLC with spectrophotometric detection at 365 nm. Sample pretreatment prior to HPLC requires little more than perchloric acid precipitation of serum or a TDO incubation mixture. The analytical recovery for the serum assay was 101 +/- 2%, while the run-to-run coefficient of variation at normal KYN levels was approximately 8%. Serum KYN levels in 40 apparently healthy fasting humans were normally distributed and ranged from 0.27 to 0.69 microgram/ml (mean +/- SD: 0.47 +/- 0.1). Serum KYN in predialysis specimens from a group of 20 patients with chronic renal failure demonstrated a highly significant increase (mean +/- SD: 0.83 +/- 0.35 microgram/ml; P less than 0.001) as compared to the reference population. It is possible that such an increase might contribute to the pathophysiology of the uremic state. The analytical recovery of KYN from TDO incubation mixtures was approximately 90%. There was no evidence for the onward metabolism of KYN during the assay of whole liver homogenates. The mean (+/- SD) TDO activity of rat liver homogenates preincubated with ascorbate and hematin was 2.3 +/- 0.8 mumol/h/g wet wt (30 degrees C). The sensitivity, specificity, and convenience of these two methods suggest that they are suitable for routine use in the investigation of the biology and pathology of oxidative tryptophan metabolism.     

Changes in kynurenine pathway metabolism in the brain, liver and kidney of aged female Wistar rats

The kynurenine pathway of tryptophan catabolism plays an important role in several biological systems affected by aging. We quantified tryptophan and its metabolites kynurenine (KYN), kynurenine acid (KYNA), picolinic acid (PIC) and quinolinic acid (QUIN), and activity of the kynurenine pathway enzymes indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO) and quinolinic acid phosphoribosyltransferase (QPRTase), in the brain, liver and kidney of young, middle-aged and old female Wistar rats. Tryptophan levels and TDO activity decreased in all tissues with age. In contrast, brain IDO activity increased with age, while liver and kidney IDO activity decreased with age. The levels of KYN, KYNA, QUIN and PIC in brain all increased with age, while the levels of KYN in the liver and kidney showed a tendency to decrease. The levels of KYNA in the liver did not change, but the levels of KYNA in the kidney increased. The levels of PIC and QUIN increased significantly in the liver but showed a tendency to decrease in the kidney. QPRTase activity in both brain and liver decreased with age but was elevated in the kidney in middle-aged (12-month-old) rats. These age-associated changes in tryptophan metabolism have the potential to impact upon major biological processes, including lymphocyte function, pyridine (NAD(P)(H)) synthesis and N-methyl-d-aspartate (NMDA)-mediated synaptic transmission, and may therefore contribute to several degenerative changes of the elderly.     

灰葡萄孢犬尿氨酸途径关键酶基因的鉴定

【背景】灰葡萄孢是一种重要的植物病原真菌,实验室前期明确了灰葡萄孢犬尿氨酸单加氧酶(kynurenine3-monooxygenase,KMO)基因BcKMO参与调控病菌的生长发育和致病力。犬尿氨酸单加氧酶(KMO)是犬尿氨酸途径的关键酶,但灰葡萄孢是否存在犬尿氨酸途径及其在病菌生长、发育和致病过程中的功能尚未见相关报道。【目的】鉴定灰葡萄孢犬尿氨酸途径中的关键酶基因,确定灰葡萄孢犬尿氨酸途径的存在,为阐明灰葡萄孢生长发育和致病力的分子机理奠定基础。【方法】利用生物信息学方法,对灰葡萄孢犬尿氨酸途径中犬尿氨酸酶(kynureninase,KYN)、吲哚-2,3-双加氧酶(indoleamine-2,3-dioxygenase,IDO)、犬尿氨酸氨基转移酶(kynurenine amino transferase,KAT)的编码基因进行分析;利用Real-time PCR技术,检测灰葡萄孢野生型BC22、BcKMO基因T-DNA插入突变体BCG183、恢复菌株BCG183/BcKMO中犬尿氨酸途径关键酶基因的表达水平;利用真菌犬尿氨酸酶KYN检测试剂盒,测定BcKMO突变体中犬尿氨酸酶(KYN)的含量。【结果】灰葡萄孢中含有2个犬尿氨酸氨基转移酶(KAT)的编码基因、3个吲哚-2,3-双加氧酶(IDO)的编码基因、10个犬尿氨酸氨基转移酶(KAT)的编码基因。灰葡萄孢KYN编码基因、IDO编码基因、KAT编码基因在突变体BCG183中的表达水平显著高于或低于在野生型和恢复菌株。突变体BCG183中犬尿氨酸酶(KYN)的含量显著低于野生型BC22和恢复菌株。【结论】灰葡萄孢中存在犬尿氨酸途径,灰葡萄孢BcKMO基因突变影响KYN、IDO和KAT编码基因的表达以及犬尿氨酸酶(KYN)的含量。     

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.     

Purification and characterization of kynurenine formamidase activities from Streptomyces parvulus

Two forms of kynurenine formamidase (EC 3.5.1.9; aryl-formylamine amidohydrolase) are present in extracts of Streptomyces parvulus. The higher molecular weight enzyme (Mr = 42 000), kynurenine formamidase I, appears to be constitutive and is present at relatively constant but low levels in antibiotic producing and nonproducing cultures, whereas the synthesis of the lower molecular weight form (Mr = 25 000), kynurenine formamidase II, is initiated just prior to the onset of actinomycin formation. It is postulated (i) that kynurenine formamidase II catalyzes the second step in the pathway from tryptophan----actinocin, and (ii) that it is regulated specifically for the specialized function of actinomycin biosynthesis. The role of kynurenine formamidase I is unknown. Formamidase I and II activities were purified from extracts of S. parvulus and kinetic parameters of the two enzymes were determined. Although some of the properties of the two enzymes are quite similar (substrate specificities, Km values), some striking differences were noted (pH and temperature optima, molecular size, chromatographic properties, sensitivity to certain ions and chemicals). Mutant studies suggest that expression of the gene(s) coding for formamidase II activity play an essential role in regulating the formation of actinocin and, hence, antibiotic synthesis. Kynurenine formamidase activity was also found in a representative number of Streptomyces species and related organisms suggesting that the enzyme may function in the degradative metabolism of tryptophan by certain actinomycetes in nature.     

Cloning and functional expression of human kynurenine 3-monooxygenase

Kynurenine 3-monooxygenase, an NADPH-dependent flavin monooxygenase, catalyses the hydroxylation of

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-kynurenine to

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-3-hydroxykynurenine. By hybridization screening using a cDNA probe encoding the entire exon 2 of Drosophila melanogaster kynurenine 3-monooxygenase, we isolated a 2.0 kb cDNA clone coding for the corresponding human liver enzyme. The deduced amino acid sequence of the human protein consists of 486 amino acids with a predicted molecular mass of 55 762 Da. Transfection of the human cDNA in HEK-293 cells resulted in the functional expression of the enzyme with kinetic properties similar to those found for the native human protein. RNA blot analysis of human tissues revealed the presence of a major mRNA species of 2.0 kb in liver, placenta and kidney.     

Biochemical and phenotypic abnormalities in kynurenine aminotransferase II-deficient mice

Kynurenic acid (KYNA) can act as an endogenous modulator of excitatory neurotransmission and has been implicated in the pathogenesis of several neurological and psychiatric diseases. To evaluate its role in the brain, we disrupted the murine gene for kynurenine aminotransferase II (KAT II), the principal enzyme responsible for the synthesis of KYNA in the rat brain. mKat-2(-/-) mice showed no detectable KAT II mRNA or protein. Total brain KAT activity and KYNA levels were reduced during the first month but returned to normal levels thereafter. In contrast, liver KAT activity and KYNA levels in mKat-2(-/-) mice were decreased by >90% throughout life, though no hepatic abnormalities were observed histologically. KYNA-associated metabolites kynurenine, 3-hydroxykynurenine, and quinolinic acid were unchanged in the brain and liver of knockout mice. mKat-2(-/-) mice began to manifest hyperactivity and abnormal motor coordination at 2 weeks of age but were indistinguishable from wild type after 1 month of age. Golgi staining of cortical and striatal neurons revealed enlarged dendritic spines and a significant increase in spine density in 3-week-old mKat-2(-/-) mice but not in 2-month-old animals. Our results show that gene targeting of mKat-2 in mice leads to early and transitory decreases in brain KAT activity and KYNA levels with commensurate behavioral and neuropathological changes and suggest that compensatory changes or ontogenic expression of another isoform may account for the normalization of KYNA levels in the adult mKat-2(-/-) brain.     

Simple separation of tritiated water and [3H]deoxyuridine from [5-3H]deoxyuridine 5'-monophosphate in the thymidylate synthase assay

A simple micromethod was developed for the accurate measurement of the activity of dTMP synthase in rat liver crude extracts. The reaction product of dTMP synthase activity assay, i.e., tritiated water, generated by the release of tritium from carbon-5 of [5-3H]deoxyuridine 5'-monophosphate (dUMP), was separated simply by 100% KOH absorption from [5-3H]deoxyuridine (dUrd), which is the side-product by dephosphorylation of [5-3H]deoxyuridine (dUrd), which is the side-product by dephosphorylation of [5-3H]dUMP during the enzyme reaction. Tritiated water was trapped in three droplets of 100% KOH deposited on the underside of the vessels' lids, while [3H]dUrd remained in the bottom of vessels after absorption of the substrate, [5-3H]dUMP, from the reaction mixture by charcoal treatment. Under standard assay conditions in the crude extract of rat liver, the specific activities of dTMP synthase and dUMP phosphatase were 0.092 +/- 0.002 and 0.351 +/- 0.013 nmol/h/mg protein, respectively. This method was also adapted for dTMP synthase assay in crude extracts of rat hepatoma 3924A. The major advantages of this procedure are the elimination of the phosphatase activity which interferes with the estimation of dTMP synthase activity in crude extracts, one-step separation of 3H2O, high sensitivity (with a limit of detection of 10 pmol of 3H2O production), high reproducibility (less than +/- 4.3%), and capability to measure activity in small amounts of sample (30-45 micrograms protein).     

Hepatic 5'-deiodinase activity of Japanese quail using reverse-T3 as substrate: assay validation, characterization, and developmental studies

Using rT3 as substrate, an in vitro 5'D assay was validated for use with liver tissue from adult Japanese quail, by defining conditions under which activity is proportional to enzyme (protein) concentration and is linear with incubation time. Activity was measured as the release of 125I from labeled rT3. Using validated assay conditions we found the following 5'D characteristics: maximal activity from 10 to 50 mM dithiothreitol (cofactor), an apparent Km of 0.52 microM rT3, pH optimum of 7.6-8.5, complete inhibition by 1 mM propylthiouracil and by 1.0 mM iopanoic acid, and substrate "preference" of rT3 greater than T4 greater than T3. Based on these characterizations the quail hepatic 5'D activity is like the Type I 5'D activity found in mammalian liver and kidney and embryonic chicken liver. To determine how previous unvalidated assays, that used high tissue and relatively low substrate (T4) concentrations, influenced 5'D studies we reevaluated 5'D development using an assay validated for each developmental stage with rT3 as substrate. We found extreme quantitative differences in the activities measured and in the proportional relationships between stages, and only limited qualitative similarity in the pattern of 5'D development when unvalidated T4 assay results were compared with validated rT3 assay results. Our data in this paper show good correspondence between whole liver 5'D activity per unit body weight and plasma T3/T4 ratios for the developmental stages sampled.     

Perinatal kynurenine pathway metabolism in the normal and asphyctic rat brain

Summary. The kynurenine pathway of tryptophan degradation contains several metabolites which may influence brain physiology and pathophysiology. The brain content of one of these compounds, kynurenic acid (KYNA), decreases precipitously around the time of birth, possibly to avoid deleterious N-methyl-D-aspartate (NMDA) receptor blockade during the perinatal period. The present study was designed to determine the levels of KYNA, the free radical generator 3-hydroxykynurenine (3-HK), and their common precursor L-kynurenine (L-KYN) between gestational day 16 and adulthood in rat brain and liver. The cerebral activities of the biosynthetic enzymes of KYNA and 3-HK, kynurenine aminotransferases (KATs) I and II and kynurenine 3-hydroxylase, respectively, were measured at the same ages. Additional studies were performed to assess whether and to what extent kynurenines in the immature brain derive from the mother, and to examine the short-term effects of birth asphyxia on brain KYNA and 3-HK levels. The results revealed that 1) the brain and liver content of L-KYN, KYNA and 3-HK is far higher pre-term than postnatally; 2) KAT I and kynurenine 3-hydroxylase activities are quite uniform between E-16 and adulthood, whereas KAT II activity rises sharply after postnatal day 14; 3) during the perinatal period, KYNA, but not L-KYN, may originate in part from the maternal circulation; and 4) oxygen deprivation at birth affects the brain content of both KYNA and 3-HK 1 h but not 24 h later. Received August 31, 1999 Accepted September 20, 1999     

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.