Structure, mechanism, and substrate specificity of kynureninase.

The kynurenine pathway is the major route for tryptophan catabolism in animals and some fungi and bacteria. The procaryotic enzyme preferentially reacts with l-kynurenine, while eucaryotic kynureninases exhibit higher activity with 3-hydroxy-l-kynurenine. Crystallography of kynureninases from Pseudomonas fluorescens (PfKyn) and Homo sapiens (HsKyn) shows that the active sites are nearly identical, except that His-102, Asn-333, and Ser-332 in HsKyn are replaced by Trp-64, Thr-282, and Gly-281 in PfKyn. Site-directed mutagenesis of HsKyn shows that these residues are, at least in part, responsible for the differences in substrate specificity since the H102W/S332G/N333T triple mutant shows activity with kynurenine but not 3-hydroxykynurenine. PfKyn is strongly inhibited by analogs of a proposed gem-diolate intermediate, dihydrokynurenine, and S-(2-aminophenyl)-l-cysteine S,S-dioxide, with K(i) values in the low nanomolar range. Stopped-flow kinetic experiments show that a transient quinonoid intermediate is formed on mixing, which decays to a ketimine at 740 s(-1). Quench experiments show that anthranilate, the first product, is formed in a stoichiometric burst at 50 s(-1) and thus the rate-determining step in the steady-state is the release of the second product, l-Ala. β-Benzoylalanine is also a good substrate for PfKyn but does not show a burst of benzoate formation, indicating that the rate-determining step for this substrate is benzoate release. A Hammett plot of rate constants for substituted β-benzoylalanines is non-linear, suggesting that carbonyl hydration is rate-determining for electron-donating groups, but C(β)-C(γ) cleavage is rate-determining for electron-withdrawing groups. This article is part of a Special Issue entitled: Pyridoxal phosphate Enzymology.