REACTION-MECHANISM OF ESCHERICHIA-COLI CYSTATHIONINE GAMMA-SYNTHASE - DIRECT EVIDENCE FOR A PYRIDOXAMINE DERIVATIVE OF VINYLGLYOXYLATE AS A KEY INTERMEDIATE IN PYRIDOXAL-PHOSPHATE DEPENDENT GAMMA-ELIMINATION AND GAMMA-REPLACEMENT REACTIONS
REACTION-MECHANISM OF ESCHERICHIA-COLI CYSTATHIONINE GAMMA-SYNTHASE - DIRECT EVIDENCE FOR A PYRIDOXAMINE DERIVATIVE OF VINYLGLYOXYLATE AS A KEY INTERMEDIATE IN PYRIDOXAL-PHOSPHATE DEPENDENT GAMMA-ELIMINATION AND GAMMA-REPLACEMENT REACTIONS
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DOI：
10.1021/bi00454a020
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发表时间：
1990-01-16
期刊：
影响因子：
2.9
通讯作者：
DUNN, MF
中科院分区：
文献类型：
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作者：
BRZOVIC, P;HOLBROOK, EL;DUNN, MF
半胱硫氨酸-γ-合酶催化O-琥珀酰基-L-高丝氨酸和L-半胱氨酸通过γ-取代反应合成依赖于磷酸吡哆醛的胱硫氨酸。在没有L半胱氨酸的情况下，OSHS经历了酶催化的伽马消除反应，生成琥珀酸、α-酮丁酸酯和氨。由于γ-取代基的消除对于两个反应都是必需的，因此可以合理地假设取代反应和消除反应路径偏离共同的中间体。以前，这种分配中间体被分配给高度共轭的α-亚胺基甘氨酸奎宁(Johnston等人，1979a)。本文报道的实验支持对分配中间体的另一种分配。我们用快速扫描停流和单波长停流紫外可见光谱研究了半胱硫醚-γ-合酶的γ-取代反应和γ-消除反应。伽马消除反应的特征是在422 nm处酶的内醛光谱带的幅度迅速下降，并伴随着在300 nm区域吸收的新物种的出现。随后，485 nm的物种以慢得多的松弛方式积累。伽马置换反应显示422 nm的峰值红移到425 nm，这发生在实验死时间(APPRX)中。3ms)。在此弛豫之后，在425 nm处的吸光度下降，这与在300 nm区域吸收的物种的外观紧密耦合。底物类似物L-丙氨酸和L-烯丙基甘氨酸与半胱硫氨酸-γ-合成酶反应，导致422 nm的吸光度漂白，并出现300 nm的物种。在没有L-半胱氨酸的情况下，L-烯丙基甘氨酸进行了简单的质子交换；在L-半胱氨酸的存在下，L-烯丙基甘氨酸发生了γ-取代反应，生成了新的氨基酸-γ-甲基胱硫氨酸。在这两个反应中，都不会积累长波吸收物质。这些结果表明，分配中间体是α-亚氨基-β，伽马-不饱和吡哆胺衍生物，其中λmax为simeq。300 nm，在消除反应中积累的485 nm物种不在替换路径上。
Cystathionine .gamma.-synthase catalyzes a pyridoxal phosphate dependent synthesis of cystathionine from O-succinyl-L-homoserine (OSHS) and L-cysteine via a .gamma.-replacement reaction. In the absence of L-cysteine, OSHS undergoes an enzyme-catalyzed, .gamma.-elimination reaction to form succinate, .alpha.-ketobutyrate, and ammonia. Since elimination of the .gamma.-substituent is necessary for both reactions, it is reasonable to assume that the replacement and elimination reaction pathways diverge from a common intermediate. Previously, this partitioning intermediate has been assigned to a highly conjugated .alpha.-iminovinylglycine quininoid (Johnston et al., 1979a). The experiments reported herein support an alternative assignment for the partitioning intermediate. We have examined the .gamma.-replacement and .gamma.-elimination reactions of cystathionine .gamma.-synthase via rapid-scanning stopped-flow and single-wavelength stopped-flow UV-visible spectroscopy. The .gamma.-elimination reaction is characterized by a rapid decrease in the amplitude of the enzyme internal aldimine spectral band at 422 nm with a concomitant appearance of a new species which absorbs in the 300-nm region. A 485-nm species subsequently accumulates in a much slower relaxation. The .gamma.-replacement reaction shows a red shift of the 422-nm peak to 425 nm which occurs in the experiment dead time (.apprx. 3 ms). This relaxation is followed by a decrease in absorbance at 425 nm that is tightly coupled to the appearance of a species which absorbs in the 300-nm region. Reaction of the substrate analogues L-alanine and L-allylglycine with cystathionine .gamma.-synthase results in bleaching of the 422-nm absorbance and the appearance of a 300-nm species. In the absence of L-cysteine, L-allylglycine undergoes facile proton exchange; in the presence of L-cysteine, L-allyglycine undergoes a .gamma.-replacement reaction to form a new amino acid, .gamma.-methylcystathionine. No long-wavelength-absorbing species accumulate during either of these reactions. These results establish that the partitioning intermediate is an .alpha.-imino .beta.,.gamma.-unsaturated pyridoxamine derivative with .lambda.max .simeq. 300 nm and that the 485-nm species which accumulates in the elimination reaction is not on the replacement pathway.