Reaction of Pseudomonas fluorescens kynureninase with beta-benzoyl-L-alanine: detection of a new reaction intermediate and a change in rate-determining step.
Beta-benzoyl-DL-alanine was synthesized from alpha-bromoacetophenone and diethyl acetamidomalonate. The racemic amino acid was resolved by carboxypeptidase A-catalyzed hydrolysis of the N-trifluoroacetyl derivative. Beta-benzoyl-L-alanine is a good substrate of kynureninase from Pseudomonas fluorescens, with k(cat) and k(cat)/K(m) values of 0.7 s(-1) and 8.0 x 10(4) M(-1) s(-1), respectively, compared to k(cat) = 16.0 s(-1) and k(cat)/K(m) = 6.0 x 10(5) M(-1) s(-1) for L-kynurenine. In contrast to the reaction of L-kynurenine, beta-benzoyl-L-alanine does not exhibit a significant solvent isotope effect on k(cat) ((H)k/(D)k = 0.96 +/- 0.06). The pre-steady-state kinetics of the reaction of beta-benzoyl-L-alanine were investigated by rapid scanning stopped-flow spectrophotometry. The spectra show the formation of a quinonoid intermediate, with lambda(max) = 490 nm, in the dead time of the instrument, which then decays, with k = 210 s(-1), to form a transient intermediate with lambda(max) at 348 nm. In the presence of benzaldehyde, the 348 nm intermediate decays, with k = 0.7 s(-1), to form a quasistable quinonoid species with lambda(max) = 492 nm. Previous studies demonstrated that benzaldehyde can trap an enamine intermediate formed after the C(beta)-C(gamma) bond cleavage [Phillips, R. S., Sundararaju, B., and Koushik, S. V. (1998) Biochemistry 37, 8783-8789]. Thus, the 348 nm intermediate is kinetically competent. The position of the absorption maximum and shape of the band is consistent with a PMP-ketimine intermediate. The results from chemical quenching analysis do not show a burst of benzoate and, thus, also support the formation of benzoate as the rate-determining step. These data suggest that, in contrast to L-kynurenine, for which the rate-determining step was shown to be deprotonation of the pyruvate-ketimine intermediate [Koushik, S. V., Moore, J. A., III, Sundararaju, B., and Phillips, R. S. (1998) Biochemistry 37, 1376-1382], the rate-determining step in the reaction of beta-benzoyl-L-alanine with kynureninase is C(beta)-C(gamma) bond cleavage.
Gawandi
Vijay B
VB
Department of Chemistry, University of Georgia, Athens, Georgia 30602-2556, USA.
Liskey
Diane
D
Lima
Santiago
S
Phillips
Robert S
RS
D000409:Alanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D001565:Benzoates / Q000737:chemistry; D006867:Hydrolases / Q000737:chemistry*; D007700:Kinetics; D007737:Kynurenine / Q000737:chemistry; D011522:Protons; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D019289:Pyruvic Acid / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013053:Spectrophotometry; D013379:Substrate Specificity
D001426:Bacterial Proteins; D001565:Benzoates; D011522:Protons; D011994:Recombinant Proteins; C047207:N-benzoylalanine; D007737:Kynurenine; D019289:Pyruvic Acid; D006867:Hydrolases; C021480:kynureninase; D000409:Alanine