Purification and biochemical characterization of some of the properties of recombinant human kynureninase

Abstract

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 (K_m = 3.0 µm ± 0.10) and was inhibited by l‐kynurenine (K_i = 20 µm), d‐kynurenine (K_i = 12 µm) and a synthetic substrate analogue d,l‐3,7‐dihydroxydesaminokynurenine (K_i = 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.