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[摘要]：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-m-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 kynureninasc. |
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