Human kynurenine aminotransferaseII-reactivity with substrates and inhibitors

• Published in: The FEBS journal 2011-06, Vol.278 (11), p.1882
• Main Authors: Passera, Elisabetta, Campanini, Barbara, Rossi, Franca, Casazza, Valentina, Rizzi, Menico, Pellicciari, Roberto, Mozzarelli, Andrea
• Format: Article
• Language: English
• Subjects: Biochemistry; Enzymes; Molecular biology; Mutation; Neurobiology; Schizophrenia
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2011.08106.x

Kynurenine aminotransferase (KAT) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the conversion of kynurenine, an intermediate of the tryptophan degradation pathway, into kynurenic acid, an endogenous antagonist of ionotropic excitatory amino acid receptors in the central nervous system. KATII is the prevalent isoform in mammalian brain and a drug target for the treatment of schizophrenia. We have carried out a spectroscopic and functional characterization of both the human wild-type KATII and a variant carrying the active site mutation Tyr142[arrow right]Phe. The transamination and the β-lytic activity of KATII towards the substrates kynurenine and α-aminoadipate, the substrate analog β-chloroalanine and the inhibitors (R)-2-amino-4-(4-(ethylsulfonyl))-4-oxobutanoic acid and cysteine sulfinate were investigated with both conventional assays and a novel continuous spectrophotometric assay. Furthermore, for high-throughput KATII inhibitor screenings, an endpoint assay suitable for 96-well plates was also developed and tested. The availability of these assays and spectroscopic analyses demonstrated that (R)-2-amino-4-(4-(ethylsulfonyl))-4-oxobutanoic acid and cysteine sulfinate, reported to be KATII inhibitors, are poor substrates that undergo slow transamination. Kynurenine aminotransferase (KAT) is a pyridoxal 5'-phosphate-dependent enzyme catalyzing the formation of kynurenic acid, an antagonist of excitatory receptors in the CNS. KATII is a potential drug target for the treatment of schizophrenia. KATII spectroscopy, transamination and β-lytic activity were characterized with substrate and substrate analogs using novel continuous and high-throughput assays. [PUBLICATION ABSTRACT]