Benzoyl-L-alanine

Kinetic and thermodynamic parameters were evaluated for the acylation and the deacylation steps in the hydrolysis of p-nitrophenyl acetate by alpha-chymotrypsin at pH 7.8 and at temperatures between 15 and 35 degrees C by the use of stopped-flow and ordinary ultraviolet spectrophotometers. In contrast to the temperature dependencies of k2 and Ks reported in the literature (P.A. Adams and E.R. Swart, Biochem. J., 161, 83 (1977], no kinetic anomaly was observed in either of the steps, but reasonable straight lines were obtained in both Arrhenius and van't Hoff plots. On the other hand, in the chymotryptic hydrolysis of N-benzoyl-L-alanine methyl ester a sharp kinetic anomaly was found. The discrepancy in the case of p-nitrophenyl acetate is discussed in connection with a possible conformational change of the enzyme, an alteration of the rate-limiting step or differences in the experimental procedures. The cause of the anomaly observed in the case of N-benzoyl-L-alanine methyl ester is also discussed in detail.

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.

FT-Raman and FT-IR spectra of N-{(meta-ferrocenyl) Benzoyl} - l-alanine - glycine ethyl ester were recorded in solid phase. The optimized molecular geometry, the vibrational wavenumbers, the infrared intensities and the Raman scattering intensities were calculated by using density functional method(B3LYP) with 6-31G(d, p) basis set. Vibrational assignment of the molecule was done by using potential energy distribution analysis. Natural bond orbital analysis, Mulliken charge analysis and HOMO-LUMO energy were used to elucidate the reasons for intra molecular charge transfer. Docking studies were conducted to predict its anticancer activity.