Kynureninase (l‐kynurenine hydrolase, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/7/1/3.html) catalyzes the hydrolytic cleavage of l‐kynurenine to l‐alanine and anthranilic acid. The proposed mechanism of the retro‐Claisen reaction requires extensive acid/base catalysis. Previous crystal structures showed that Tyr226 in the Pseudomonas fluorescens enzyme (Tyr275 in the human enzyme) hydrogen bonds to the phosphate of the pyridoxal‐5′‐phosphate (PLP) cofactor. This Tyr residue is strictly conserved in all sequences of kynureninase. The human enzyme complexed with a competitive inhibitor, 3‐hydroxyhippuric acid, showed that the ligand carbonyl O is located 3.7 Å from the phenol of Tyr275 (Lima, S., Kumar, S., Gawandi, V., Momany, C. & Phillips, R. S. (2009) J. Med. Chem. 52, 389–396). We prepared a Y226F mutant of P. fluorescens kynureninase to probe the role of this residue in catalysis. The Y226F mutant has approximately 3000‐fold lower activity than wild‐type, and does not show the pKa values of 6.8 on kcat and 6.5 and 8.8 on kcat/Km seen for the wild‐type enzyme (Koushik, S. V., Moore, J. A. III, Sundararaju, B. & Phillips, R. S. (1998) Biochemistry 37, 1376–1382). Wild‐type kynureninase shows a resonance at 4.5 ppm in 31P‐NMR, which is shifted to 5.0, 3.3 and 2.0 ppm when the potent inhibitor 5‐bromodihydrokynurenine is added. However, Y226F kynureninase shows resonances at 3.6 and 2.5 ppm, and no change in the peak position is seen when 5‐bromodihydrokynurenine is added. Taken together, these results suggest that Tyr226 mediates proton transfer between the substrate and the phosphate, which accelerates formation of external aldimine and gem‐diol intermediates. Thus, the phosphate of PLP acts as an acid/base catalyst in the mechanism of kynureninase.
