Characterization of ligand-induced states of maize homoserine dehydrogenase.
The threonine-sensitive homoserine dehydrogenase (L-homoserine: NAD(P)+ oxido-reductase), isolated from seedlings of Zea mays L., is characterized by variable kinetic and regulatory properties. Previous analysis of this enzyme suggested that it is capable of ligand-mediated interconversions among four kinetically distinct states (S. Krishnaswamy and J. K. Bryan (1983) Arch. Biochem. Biophys. 222, 449-463). These forms of the enzyme have been identified and found to differ in oligomeric configuration and conformation. In the presence of KCl and threonine a rapid equilibrium among three species of the enzyme (B, T, and K) is established. Each of these species can undergo a unique slow transition to a steady-state form under assay conditions. Results obtained from gel-filtration chromatography and sucrose density centrifugation indicate that the B and steady-state forms are tetramers and the T and K states are dimers. Evidence is presented to indicate that the rapid conversion from one dimeric species to the other can only occur via formation of the tetrameric B state. Chromatography under reacting-enzyme conditions provides direct support for the slow formation of a common steady-state species from any one of the other forms of the enzyme. The rate of transition is influenced by threonine, homoserine, NAD+, and, for transitions involving association reactions, by enzyme concentration. Small, reproducible differences in the apparent size of the T and K forms, and the B and steady-state species, are attributed to changes in conformation. This conclusion is supported by differential susceptibility of the enzymic states to proteolytic inactivation, by different rates of inactivation by dithio-bis-nitrobenzoate, and by alterations in their thermal stability. In addition, the B, T, and K states of the enzyme exhibit unique intrinsic fluorescence spectra. Spectral changes are shown to closely parallel changes in kinetic and hysteretic properties of the enzyme. The results of diverse methods of analysis are internally consistent, and provide considerable support for the conclusion that this pleiotropic regulatory enzyme can exist in any of several physically distinct states.
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis*; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D006715:Homoserine Dehydrogenase / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D007700:Kinetics; D008024:Ligands / Q000494:pharmacology*; D008970:Molecular Weight; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013696:Temperature; D003313:Zea mays / Q000201:enzymology*
D008024:Ligands; D013438:Sulfhydryl Compounds; D000429:Alcohol Oxidoreductases; D006715:Homoserine Dehydrogenase