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Thermal stability, pH dependence and inhibition of four murine kynurenine aminotransferases.

ABSTRACT: Kynurenine aminotransferase (KAT) catalyzes the transamination of kynunrenine to kynurenic acid (KYNA). KYNA is a neuroactive compound and functions as an antagonist of alpha7-nicotinic acetylcholine receptors and is the only known endogenous antagonist of N-methyl-D-aspartate receptors. Four KAT enzymes, KAT I/glutamine transaminase K/cysteine conjugate beta-lyase 1, KAT II/aminoadipate aminotransferase, KAT III/cysteine conjugate beta-lyase 2, and KAT IV/glutamic-oxaloacetic transaminase 2/mitochondrial aspartate aminotransferase, have been reported in mammalian brains. Because of the substrate overlap of the four KAT enzymes, it is difficult to assay the specific activity of each KAT in animal brains.This study concerns the functional expression and comparative characterization of KAT I, II, III, and IV from mice. At the applied test conditions, equimolar tryptophan with kynurenine significantly inhibited only mouse KAT I and IV, equimolar methionine inhibited only mouse KAT III and equimolar aspartate inhibited only mouse KAT IV. The activity of mouse KAT II was not significantly inhibited by any proteinogenic amino acids at equimolar concentrations. pH optima, temperature preferences of four KATs were also tested in this study. Midpoint temperatures of the protein melting, half life values at 65 degrees C, and pKa values of mouse KAT I, II, III, and IV were 69.8, 65.9, 64.8 and 66.5 degrees C; 69.7, 27.4, 3.9 and 6.5 min; pH 7.6, 5.7, 8.7 and 6.9, respectively.The characteristics reported here could be used to develop specific assay methods for each of the four murine KATs. These specific assays could be used to identify which KAT is affected in mouse models for research and to develop small molecule drugs for prevention and treatment of KAT-involved human diseases.

SUBMITTER: Han Q

PROVIDER: S-EPMC2890522 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Thermal stability, pH dependence and inhibition of four murine kynurenine aminotransferases.

Han Qian Q Cai Tao T Tagle Danilo A DA Li Jianyong J

BMC biochemistry 20100519

<h4>Background</h4>Kynurenine aminotransferase (KAT) catalyzes the transamination of kynunrenine to kynurenic acid (KYNA). KYNA is a neuroactive compound and functions as an antagonist of alpha7-nicotinic acetylcholine receptors and is the only known endogenous antagonist of N-methyl-D-aspartate receptors. Four KAT enzymes, KAT I/glutamine transaminase K/cysteine conjugate beta-lyase 1, KAT II/aminoadipate aminotransferase, KAT III/cysteine conjugate beta-lyase 2, and KAT IV/glutamic-oxaloacetic ...[more]

PMID: 20482848

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Project description:BackgroundComplex interaction of genetic defects with environmental factors seems to play a substantial role in the pathogenesis of inflammatory bowel disease (IBD). Accumulating data implicate a potential role of disturbed tryptophan metabolism in IBD. Kynurenic acid (KYNA), a derivative of tryptophan (TRP) along the kynurenine (KYN) pathway, displays cytoprotective and immunomodulating properties, whereas 3-OH-KYN is a cytotoxic compound, generating free radicals.MethodsThe expression of lymphocytic mRNA encoding enzymes synthesizing KYNA (KAT I-III) and serum levels of TRP and its metabolites were evaluated in 55 patients with IBD, during remission or relapse [27 patients with ulcerative colitis (UC) and 28 patients with Crohn's disease (CD)] and in 50 control individuals.ResultsThe increased expression of KAT1 and KAT3 mRNA characterized the entire cohorts of patients with UC and CD, as well as relapse-remission subsets. Expression of KAT2 mRNA was enhanced in patients with UC and in patients with CD in remission. In the entire cohorts of UC or CD, TRP levels were lower, whereas KYN, KYNA and 3-OH-KYN were not altered. When analysed in subsets of patients with UC and CD (active phase-remission), KYNA level was significantly lower during remission than relapse, yet not versus control. Functionally, in the whole groups of patients with UC or CD, the TRP/KYN ratio has been lower than control, whereas KYN/KYNA and KYNA/3-OH-KYN ratios were not altered. The ratio KYN/3-OH-KYN increased approximately two-fold among all patients with CD; furthermore, patients with CD with relapse, manifested a significantly higher KYNA/3-OH-KYN ratio than patients in remission.ConclusionThe presented data indicate that IBD is associated with an enhanced expression of genes encoding KYNA biosynthetic enzymes in lymphocytes; however, additional mechanisms appear to influence KYNA levels. Higher metabolic conversion of serum TRP in IBD seems to be followed by the functional shift of KYN pathway towards the arm producing KYNA during exacerbation. We propose that KYNA, possibly via interaction with aryl hydrocarbon receptor or G-protein-coupled orphan receptor 35, may serve as a counter-regulatory mechanism, decreasing cytotoxicity and inflammation in IBD. Further longitudinal studies evaluating the individual dynamics of TRP and KYN pathway in patients with IBD, as well as the nature of precise mechanisms regulating KYNA synthesis, should be helpful in better understanding the processes underlying the observed changes.

Project description:Cinnamaldehyde, cinnamyl alcohol, β-methylstyrene and cinnamic acid are four important biomass 3-phenyl-2-propene compounds. In the field of perfume and organic synthesis, their thermal stability and oxidation pathways deserve attention. This paper reports a new attempt to investigate the thermal stability and reactivity by a custom-designed mini closed pressure vessel test (MCPVT). The pressure and temperature behaviors were measured by MCPVT under nitrogen and oxygen atmosphere. The temperature of initial oxygen absorption (T a) and rapid oxidation (T R) were calculated. The results showed that four 3-phenyl-2-propene compounds were stable under nitrogen atmosphere. The T a of cinnamaldehyde, cinnamyl alcohol, β-methylstyrene, and cinnamic acid was 271.25 K, 292.375 K, 323.125 K, and 363.875 K, and their T R was 301.125 K, 332.75 K, 357.91 K, and 385.375 K, respectively. The oxidation reactivity order was derived to be cinnamaldehyde > cinnamyl alcohol > β-methylstyrene > cinnamic acid. The oxidation kinetics were determined using n versus time (n-t) plots, which showed a second-order reaction. Peroxide was determined by iodimetry, and the oxidation products were analyzed by gas chromatography-mass spectrometry (GC-MS). The results showed that the peroxide value of cinnamaldehyde, cinnamyl alcohol, β-methylstyrene, and cinnamic acid reached 18.88, 15.07, 9.62, and 4.24 mmol kg-1 at 373 K for 6 h, respectively. The common oxidation products of four 3-phenyl-2-propene compounds were benzaldehyde, benzoic acid, and epoxide, which resulted from the carbon-carbon double bond oxidation. The substituents' oxidation products were obtained from the oxidation of cinnamaldehyde, cinnamyl alcohol, and β-methylstyrene. In particular, the difference is that no oxidation products of the carboxyl group of cinnamic acid were detected. The common oxidation products of the four 3-phenyl-2-propene compounds were benzaldehyde, benzoic acid, and epoxide, which resulted from the carbon-carbon double bond oxidation. The substituents' oxidation products were caught in the oxidation of cinnamaldehyde, cinnamyl alcohol, and β-methylstyrene. In particular, the difference is that no oxidation products of the carboxyl group of cinnamic acid were detected. According to the complex oxidation products, important insights into the oxidation pathways were provided.

Dataset Information

Thermal stability, pH dependence and inhibition of four murine kynurenine aminotransferases.

Publications

Thermal stability, pH dependence and inhibition of four murine kynurenine aminotransferases.

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