Homoserine Dehydrsgenase of Rhodospirillum rubrum

Homoserine dehydrogenase of Rhodospirillum rubrum has been purified by heat treatment, ammonium sulfate fractionation, and by two successive gel filtration steps on Sephadex G-ZOO in the absence and presence of L-threonine, an allosteric inhibitor. The pure protein is free of aspartokinase and aspartate /?-semialdehyde dehydrogenase activities. From the sedimentation velocity centrifugation and Sephadex gel filtration experiments, a molecular weight of approximately 138,000 can be calculated. Since the homoserine dehydrogenase of R. rubrum shows reversible associationdissociation behavior as well as drastic conformational alterations in the presence of various ligands, this value of 138,000 may reflect the average molecular weight of more than one form of the enzyme because of a rapid equilibrium between these species in solution. In the presence of sodium dodecyl sulfate and /3-mercaptoethanol, the native enzyme is dissociated to smaller units of approximate molecular weight of 48,000 + 5,000. Titration of the native protein with 5,5’-dithiobis(Z-nitrobenzoic acid) reveals 1 mole of -SH group per mole of enzyme; however, under denaturating conditions, between 3 and 4 moles of -SH per 140,000 g are titratable. The enzyme shows aggregation behavior in the presence of L-threonine, and the state of aggregation is dependent on the presence and absence of reducing agents and on the protein concentration. In phosphate buffer, pH 6.8, containing 0.001 M L-threonine and 0.002 M dithiothreitol, the enzyme sediments at a calculated szo,W value of 9.3 S; upon removal of threonine this high molecular weight species dissociates to its original size with an ~~0,~ value of 6.9 S. In the absence of any thiol reagent, however, a large number of catalytically active nonspecific aggregates, ranging in molecular weights from 300,000 to 2 X 106, are detected by various analytical methods such as gel filtration and polyacrylamide gel electrophoresis. A possible explanation for the formation of these aggregates is that the enzyme molecules are covalently linked through interchain disulfide bonds.