Purification and biochemical characterization of some of the properties of recombinant human kynureninase.
Recombinant human kynureninase (L-kynurenine hydrolase, EC 3.7.1.3) was purified to homogeneity (60-fold) from Spodoptera frugiperda (Sf9) cells infected with baculovirus containing the kynureninase gene. The purification protocol comprised ammonium sulfate precipitation and several chromatographic steps, including DEAE-Sepharose CL-6B, hydroxyapatite, strong anionic and cationic separations. The purity of the enzyme was determined by SDS/PAGE, and the molecular mass verified by MALDI-TOF MS. The monomeric molecular mass of 52.4 kDa determined was > 99.99% of the predicted molecular mass. A UV absorption spectrum of the holoenzyme resulted in a peak at 432 nm. The optimum pH was 8.25 and the enzyme displayed a strong dependence on the ionic strength of the buffer for optimum activity. This cloned enzyme was highly specific for 3-hydroxykynurenine (Km = 3.0 microm +/- 0.10) and was inhibited by L-kynurenine (Ki = 20 microm), d-kynurenine (Ki = 12 microm) and a synthetic substrate analogue D,L-3,7-dihydroxydesaminokynurenine (Ki = 100 nm). The activity/concentration profile for kynureninase from this source was sigmoidal in all instances. There appeared to be partial inhibition by substrate, and excess pyridoxal 5'-phosphate was found to be inhibitory.
Walsh
Harold A
HA
School of Chemistry, University of St Andrews, St Andrews, Fife, Scotland, UK.
Botting
Nigel P
NP
D003001:Cloning, Molecular; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D011732:Pyridoxal Phosphate / Q000502:physiology; D013379:Substrate Specificity / Q000502:physiology
D011732:Pyridoxal Phosphate; D006867:Hydrolases; C021480:kynureninase