Gawandi Vijay B, Liskey Diane, Lima Santiago, Phillips Robert S
Department of Chemistry, University of Georgia, Athens, Georgia 30602-2556, USA.
Biochemistry. 2004 Mar 23;43(11):3230-7. doi: 10.1021/bi036043k.
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.
β-苯甲酰基-DL-丙氨酸由α-溴代苯乙酮和二乙酰胺基丙二酸二乙酯合成。外消旋氨基酸通过羧肽酶A催化水解N-三氟乙酰基衍生物进行拆分。β-苯甲酰基-L-丙氨酸是荧光假单胞菌犬尿氨酸酶的良好底物，其k(cat)和k(cat)/K(m)值分别为0.7 s(-1)和8.0×10(4) M(-1) s(-1)，而L-犬尿氨酸的k(cat) = 16.0 s(-1)，k(cat)/K(m) = 6.0×10(5) M(-1) s(-1)。与L-犬尿氨酸的反应不同，β-苯甲酰基-L-丙氨酸对k(cat)没有显著的溶剂同位素效应（(H)k/(D)k = 0.96±0.06）。通过快速扫描停流分光光度法研究了β-苯甲酰基-L-丙氨酸反应的预稳态动力学。光谱显示在仪器的死时间内形成了一种醌型中间体，λ(max) = 490 nm，然后以k = 210 s(-1)的速率衰减，形成一个λ(max)为348 nm的瞬态中间体。在苯甲醛存在下，348 nm的中间体以k = 0.7 s(-1)的速率衰减，形成一种准稳定的醌型物种，λ(max) = 492 nm。先前的研究表明，苯甲醛可以捕获C(β)-C(γ)键断裂后形成的烯胺中间体[菲利普斯，R.S.，桑达拉朱，B.，和库希克，S.V.（1998年）《生物化学》37，8783 - 8789]。因此，348 nm的中间体在动力学上是有效的。吸收最大值的位置和谱带形状与PMP-酮亚胺中间体一致。化学淬灭分析的结果没有显示出苯甲酸的爆发，因此也支持苯甲酸的形成是速率决定步骤。这些数据表明，与L-犬尿氨酸不同，其速率决定步骤被证明是丙酮酸-酮亚胺中间体的去质子化[库希克，S.V.，摩尔，J.A.，III，桑达拉朱，B.，和菲利普斯，R.S.（1998年）《生物化学》37，1376 - 1382]，β-苯甲酰基-L-丙氨酸与犬尿氨酸酶反应的速率决定步骤是C(β)-C(γ)键断裂。
