Regulation of a metabolic system in vitro: synthesis of threonine from aspartic acid.
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.
D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006715:Homoserine Dehydrogenase / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D008190:Lyases / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism; D013912:Threonine / Q000096:biosynthesis*
D007527:Isoenzymes; D009097:Multienzyme Complexes; D013912:Threonine; D001224:Aspartic Acid; D006715:Homoserine Dehydrogenase; D000445:Aldehyde Oxidoreductases; D010770:Phosphotransferases; D008190:Lyases