Substituent effects on the reaction of beta-benzoylalanines with Pseudomonas fluorescens kynureninase

Abstract

Kynureninase is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the hydrolytic cleavage of l-kynurenine to give l-alanine and anthranilic acid. beta-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 beta-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 beta-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 C(beta)-C(gamma) 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 beta,beta-dideuterio-beta-(4-trifluoromethylbenzoyl)-dl-alanine, the 348 nm intermediate is proposed to be a vinylogous amide derived from abortive beta-deprotonation of the ketimine intermediate. These results provide additional evidence for a gem-diol intermediate in the catalytic mechanism of kynureninase.