title: Structure, mechanism, and substrate specificity of kynureninase.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Georgia, Athens, GA 30602, USA. rsphillips@chem.uga.edu
authors.forename: Robert S
authors.initials: RS
authors.lastname: Phillips
mesh_terms: D000595:Amino Acid Sequence; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011551:Pseudomonas fluorescens / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D006867:Hydrolases; C021480:kynureninase