Szczesiul M, Wampler D E
Biochemistry. 1976 May 18;15(10):2236-44. doi: 10.1021/bi00655a033.
Six enzymes involved in the conversion of aspartate to threonine have been extracted from Escherichia coli and separated from each other. Two of these enzymes, aspartokinase and homoserine dehydrogenase, have also been partially purified from Rhodopseudomonas spheroides. In an attempt to determine whether small changes in the kinetic properties of individual enzymes are important to the regulation of metabolic flux through a coupled reaction system, the partially purified enzymes were recombined in a variety of ways under reaction conditions designed to resemble the in vivo situation. These conditions include: use of an entire metabolic system rather than a single reaction; high enzyme concentrations at the same relative concentrations as found in the cell; and low, steady-state concentrations of substrates and products. Metabolic flux was followed spectrophotometrically and the concentrations of aspartic semialdehyde, hemoserine, O-phosphohomoserine, and threonine were measured. The results indicate that the threonine concentration is of major importance in regulating metabolic flux by inhibiting aspartokinase, the first reaction in threonine in the pathway. When threonine-insensitive aspartokinases were used, concentrations reached higher levels and the rate of NADPH oxidation remained higher. The fact that neither aspartic semialdehyde nor homoserine accumulated as the threonine concentration increased and the lack of correlation between changes in metabolic flux and ADP/ATP or NADPH/NADP ratios indicate that more subtle forms of metabolic regulation, such as "reverse cascade", secondary feedback sites, or "energy charge", are of little regulatory importance in this isolated, metabolic system. The results also emphasize the need for caution in projecting in vivo control mechanisms from in vitro experiments.
已从大肠杆菌中提取出参与天冬氨酸转化为苏氨酸过程的六种酶，并将它们彼此分离。其中两种酶，即天冬氨酸激酶和高丝氨酸脱氢酶，也已从球形红假单胞菌中部分纯化出来。为了确定单个酶动力学性质的微小变化对通过偶联反应系统调节代谢通量是否重要，在旨在模拟体内情况的反应条件下，以多种方式将部分纯化的酶重新组合。这些条件包括：使用整个代谢系统而非单一反应；酶浓度与细胞内发现的相对浓度相同且较高；底物和产物的浓度处于低稳态。通过分光光度法跟踪代谢通量，并测量了天冬氨酸半醛、高丝氨酸、O-磷酸高丝氨酸和苏氨酸的浓度。结果表明，苏氨酸浓度在通过抑制天冬氨酸激酶（苏氨酸途径中的第一个反应）来调节代谢通量方面至关重要。当使用对苏氨酸不敏感的天冬氨酸激酶时，浓度达到更高水平，并且NADPH氧化速率保持较高。随着苏氨酸浓度增加，天冬氨酸半醛和高丝氨酸均未积累，以及代谢通量变化与ADP/ATP或NADPH/NADP比率之间缺乏相关性，这一事实表明，在这个孤立的代谢系统中，更微妙的代谢调节形式，如“反向级联”、二级反馈位点或“能荷”，在调节方面作用不大。结果还强调了在从体外实验推断体内控制机制时需要谨慎。
