Brzović P, Holbrook E L, Greene R C, Dunn M F
Department of Biochemistry, University of California, Riverside 92521.
Biochemistry. 1990 Jan 16;29(2):442-51. doi: 10.1021/bi00454a020.
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 (approximately 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-allylglycine 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 congruent to 300 nm and that the 485-nm species which accumulates in the elimination reaction is not on the replacement pathway.
胱硫醚γ-合酶催化磷酸吡哆醛依赖性反应，通过γ-取代反应，由O-琥珀酰-L-高丝氨酸（OSHS）和L-半胱氨酸合成胱硫醚。在没有L-半胱氨酸的情况下，OSHS会发生酶催化的γ-消除反应，生成琥珀酸、α-酮丁酸和氨。由于这两个反应都需要消除γ-取代基，因此可以合理推测取代反应和消除反应途径是从一个共同的中间体分岔而来的。此前，这个分配中间体被认为是一种高度共轭的α-亚氨基乙烯基甘氨酸醌型化合物（约翰斯顿等人，1979a）。本文报道的实验支持了对分配中间体的另一种认定。我们通过快速扫描停流和单波长停流紫外可见光谱法研究了胱硫醚γ-合酶的γ-取代和γ-消除反应。γ-消除反应的特征是酶内部醛亚胺光谱带在422nm处的振幅迅速下降，同时出现一个在300nm区域有吸收的新物种。随后，一个485nm的物种以慢得多的弛豫速率积累。γ-取代反应显示422nm处的峰红移至425nm，这发生在实验死时间（约3毫秒）内。这种弛豫之后是425nm处吸光度的下降，这与一个在300nm区域有吸收的物种的出现紧密相关。底物类似物L-丙氨酸和L-烯丙基甘氨酸与胱硫醚γ-合酶反应会导致422nm处的吸光度漂白，并出现一个300nm的物种。在没有L-半胱氨酸的情况下，L-烯丙基甘氨酸容易发生质子交换；在有L-半胱氨酸的情况下，L-烯丙基甘氨酸会发生γ-取代反应，形成一种新的氨基酸，γ-甲基胱硫醚。在这两个反应过程中都没有积累长波长吸收物种。这些结果表明，分配中间体是一种α-亚氨基β,γ-不饱和吡哆胺衍生物，其最大吸收波长约为300nm，并且在消除反应中积累的485nm物种不在取代反应途径上。
