Cysteine and keto acids modulate mosquito kynurenine aminotransferase catalyzed kynurenic acid production

Abstract

Kynurenine aminotransferase (KAT) catalyzes the formation of kynurenic acid (KYNA), the natural antagonist of ionotropic glutamate receptors. This study tests potential substrates and assesses the effects of amino acids and keto acids on the activity of mosquito KAT. Various keto acids, when simultaneously present in the same reaction mixture, display a combined effect on KAT catalyzed KYNA production. Moreover, methionine and glutamine show inhibitory effects on KAT activity, while cysteine functions as either an antagonist or an inhibitor depending on the concentration. Therefore, the overall level of keto acids and cysteine might modulate the KYNA synthesis. Results from this study will be useful in the study of KAT regulation in other animals.

Fig. 1

Cosubstrate specificity of AeKAT. AeKAT was incubated in the presence of 15 mM kynurenine and 0.16–20 mM different keto acids. The activity was quantitated by the amount of KYNA produced in the reaction mixture. The units are labeled at the right corner.

Fig. 2

Effect of other amino acids on enzyme activity. Assay conditions were similar to those described in Section 2.2, except that kynurenine and ketobutyrate concentrations were 3 and 2 mM, respectively. The concentration of the additional amino acid incorporated into the reaction mixture was 2 mM. The activity was quantitated by the amount of KYNA produced in the reaction mixture. The star labeled bars are significantly different from that of the control without additional amino acid. *P < 0:5, **P < 0:01.

Fig. 3

Effect of the multiple keto acids on AeKAT-catalyzed KYNA production. (a) Rate of KYNA production in the AeKAT and kynurenine mixture in the presence of 3 mM of ketobutyrate and 2 mM of a different keto acids. The star labeled bars are significantly different from the bar for the control without adding any other keto acid. *P < 0:5, **P < 0:01. (b) Inhibition of KYNA production in the Ae-KAT/kynurenine mixture in the presence of three keto acids, αKMB, glyoxylate, and p-hydroxy-phenylpyruvate. They were used either simultaneously at 2 mM each (combination) or 2 mM any one of them alone. (c) Enhancement of KYNA production by AeKAT in the presence of three different keto acids, pyruvate, ketoglutarate and oxaloacetate. They were used either simultaneously at 1 mM each (combination) or 1 mM any one of them alone. HPP, p-hydroxy-phenylpyruvate.

Fig. 4

Effect of cysteine on enzyme activity. Assay conditions were similar to those described in Section 2.2. The activity was calculated by the amount of KYNA produced in the reaction mixture. The bars labeled with stars are significantly different from the bar for the control without adding any cysteine. *P < 0:5, **P < 0:01.