Stereospecificity of Pseudomonas fluorescens kynureninase for diastereomers of beta-methylkynurenine.
The diastereomers of beta-methyl-L-kynurenine were prepared by preparative ozonolysis of the respective diastereomers of beta-methyl-L-tryptophan. A practical method for preparative enzymatic resolution of the diastereomers of beta-methyltryptophan was developed using carboxypeptidase A digestion of the N-trifluoroacetyl derivatives. The stereochemical assignment was confirmed by X-ray crystal structure determination of (2S, 3R)-threo-beta-methyl-L-tryptophan. (2S,3S)-erythro-beta-Methyl-L-kynurenine is a slow substrate for kynureninase from Pseudomonas fluorescens (k(cat)/K(m) = 0.1% that of L-kynurenine), producing anthranilic acid, while (2S,3R)-threo-L-kynurenine is about 390-fold less reactive than erythro. Rapid-scanning stopped-flow measurements show that beta-methyl substitution affects the rate of alpha-deprotonation of the L-kynurenine-pyridoxal-5'-phosphate Schiffs base. This is consistent with the stereoelectronic requirements of the reaction. These results are the first demonstration that beta-substituted kynurenines can be substrates for kynureninase, and may be useful in the design of mechanism-based inhibitors.
Cyr
L V
LV
Department of Chemistry, Center for Metalloenzyme Studies, University of Georgia, Athens 30602-2556, USA.
Newton
M G
MG
Phillips
R S
RS
D006867:Hydrolases / Q000378:metabolism*; D007700:Kinetics; D007737:Kynurenine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity
C121897:beta-methylkynurenine; D007737:Kynurenine; D006867:Hydrolases; C021480:kynureninase