Substituent Effects on the Reaction of β-Benzoylalanines with Pseudomonas fluorescens Kynureninase

• Published in: Biochemistry (Easton) 2010-09, Vol.49 (36), p.7913-7919
• Main Authors: Kumar, Sunil, Gawandi, Vijay B, Capito, Nicholas, Phillips, Robert S
• Format: Article
• Language: English
• Subjects: Alanine - analogs & derivatives; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Catalysis; Hydrolases - chemistry; Hydrolases - metabolism; Kinetics; Kynurenine - analogs & derivatives; Kynurenine - chemistry; Pseudomonas fluorescens - enzymology; Pseudomonas fluorescens - metabolism; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi100955b

Kynureninase is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the hydrolytic cleavage of l-kynurenine to give l-alanine and anthranilic acid. β-Benzoyl-l-alanine, the analogue of l-kynurenine lacking the aromatic amino group, was shown to a good substrate for kynureninase from Pseudomonas fluorescens, and the rate-determining step changes from release of the second product, l-Ala, to formation of the first product, benzoate [Gawandi, V. B., et al. (2004) Biochemistry 43, 3230−3237]. In this work, a series of aryl-substituted β-benzoyl-dl-alanines was synthesized and evaluated for substrate activity with kynureninase from P. fluorescens. Hammett analysis of k cat and k cat/K m for 4-substituted β-benzoyl-dl-alanines with electron-withdrawing and electron-donating substituents is nonlinear, with a concave downward curvature. This suggests that there is a change in rate-determining step for benzoate formation with different substituents, from gem-diol formation for electron-donating substituents to Cβ−Cγ bond cleavage for electron-withdrawing substituents. Rapid-scanning stopped-flow kinetic experiments demonstrated that substituents have relatively minor effects on formation of the quinonoid and 348 nm intermediates but have a much greater effect on the formation of the aldol product from reaction of benzaldehyde with the 348 nm intermediate. Since there is a kinetic isotope effect on its formation from β,β-dideuterio-β-(4-trifluoromethylbenzoyl)-dl-alanine, the 348 nm intermediate is proposed to be a vinylogous amide derived from abortive β-deprotonation of the ketimine intermediate. These results provide additional evidence for a gem-diol intermediate in the catalytic mechanism of kynureninase.