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

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Effect of temperature on enzyme activity. The activities of recombinant mKATs at different temperatures. a) mKAT I and mKAT II, b) mKAT III and mKAT IV.

Figure 2

The absorbance changes at 280 nm of four mKAT proteins after heat treatment. a, mKAT I; b, mKAT II; c, mKAT III, and d, mKAT IV.

Figure 3

Thermal stability of four mKAT proteins at 65°C. a, Decay curves of mKAT I and mKAT II; b, mKAT III and mKAT IV.

Figure 4

Effect of pH on enzyme activity. The activities of recombinant mKATs at different pH values. a) mKAT I, b) mKAT II, c) mKAT III, d) mKAT IV.

Figure 5

Transamination activity of mKAT IV towards different amino acids with α-ketoglutarate or oxaloacetate as an amino group acceptor. The activity was quantified by the amount of glutamate or aspartate produced in the reaction mixture. Oxaloacetate was only used for testing KAT activity with glutamate. 3-HK, 3-hydroxy-DL-kynurenine, Kyn: kynurenine.

Figure 6

Effect of other amino acids on mKAT enzyme activities. The activities were quantified by the amount of KYNA produced in the reaction mixtures in 100 mM phosphate, pH 7.5. The mixture contains 5 mM kynurenine, 2 mM α-ketoglutarate (for mKAT I and III) or glyoxylate (for mKAT I and III), 5 mM other amino acid. The rate of KYNA production for each mKAT and different reaction mixture is shown in the figure. The bars labeled with stars (**P < 0.01, * P < 0.05) are significant different from the control (Kyn). Kyn: kynurenine, 3-HK, 3-hydroxy-DL-kynurenine. a) mKAT I, b) mKAT II, c) mKAT III, d) mKAT IV.

Figure 7

Transamination activity of mouse brain crude protein extract towards different α-ketoacids. The reaction mixture consists of 5 mM kynurenine, 2 mM α-keto acid, 40 μM PLP, 20 μg brain crude protein extract in 100 μL 100 mM phosphate buffer, pH 7.5. The mixture was incubated at 38°C for 2 h and the reaction was stopped by adding an equal volume of 0.8 M formic acid. Measurement of KYNA was performed by HPLC with fluorometric detection at Exc. 340 nm and Em. 398 nm.

Figure 8

Effect of four other amino acids on KAT activity of mouse brain crude protein extract. The reaction mixture consists of 5 mM kynurenine, 2 mM co-substrate (glyoxylate or α-ketoglutarate), 40 μM PLP, 5 mM other amino acid (putative inhibitor), 20 μg brain crude protein extract in 100 μL 100 mM phosphate buffer, pH 7.5 or 100 mM sodium borate buffer, pH 9. The mixture was incubated at 38°C for 2 h. The reaction was stopped by adding an equal volume of 0.8 M formic acid. Measurement of KYNA was performed by HPLC with fluorometric detection at Exc. 340 nm and Em. 398 nm. a) Results at pH 7.5 using α-ketoglutarate as a co-substrate, b) Results at pH 9 using glyoxylate as a co-substrate.