Characteristic features of kynurenine aminotransferase allosterically regulated by (alpha)-ketoglutarate in cooperation with kynurenine.
Kynurenine aminotransferase from Pyrococcus horikoshii OT3 (PhKAT), which is a homodimeric protein, catalyzes the conversion of kynurenine (KYN) to kynurenic acid (KYNA). We analyzed the transaminase reaction mechanisms of this protein with pyridoxal-5'-phosphate (PLP), KYN and α-ketoglutaric acid (2OG) or oxaloacetic acid (OXA). 2OG significantly inhibited KAT activities in kinetic analyses, suggesting that a KYNA biosynthesis is allosterically regulated by 2OG. Its inhibitions evidently were unlocked by KYN. 2OG and KYN functioned as an inhibitor and activator in response to changes in the concentrations of KYN and 2OG, respectively. The affinities of one subunit for PLP or 2OG were different from that of the other subunit, as confirmed by spectrophotometry and isothermal titration calorimetry, suggesting that the difference of affinities between subunits might play a role in regulations of the KAT reaction. Moreover, we identified two active and allosteric sites in the crystal structure of PhKAT-2OG complexes. The crystal structure of PhKAT in complex with four 2OGs demonstrates that two 2OGs in allosteric sites are effector molecules which inhibit the KYNA productions. Thus, the combined data lead to the conclusion that PhKAT probably is regulated by allosteric control machineries, with 2OG as the allosteric inhibitor.
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Osaka, Japan. okadak@mls.eng.osaka-u.ac.jp
Ken
K
Okada
Clement
C
Angkawidjaja
Yuichi
Y
Koga
Kazufumi
K
Takano
Shigenori
S
Kanaya
D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002151:Calorimetry; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006801:Humans; D007656:Ketoglutaric Acids / Q000737:chemistry*; D007700:Kinetics; D007737:Kynurenine / Q000737:chemistry*; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011732:Pyridoxal Phosphate / Q000737:chemistry; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D040681:Structural Homology, Protein; D013499:Surface Properties; D013816:Thermodynamics; D000637:Transaminases / Q000737:chemistry*
D007656:Ketoglutaric Acids; D007737:Kynurenine; D011732:Pyridoxal Phosphate; D000637:Transaminases; C021481:kynurenine-oxoglutarate transaminase