ABSTRACT
A detailed physicochemical characterization of purified homoserine dehydrogenase of Rhodospirillum rubrum is presented. The enzyme has a molecular weight of 110000 and consists of two subunits of identical molecular weight of 55000. Depending on the ionic strength and protein concentration it is possible for the native enzyme to dimerize to produce an enzymatically active species of molecular weight 220000. Titrations of the native and detergent-treated enzyme with a variety of sulfhydryl reagents show 2 mol free--SH groups per 110000 g, one of which is buried in the protein interior. L-Threonine and/or high concentrations of salt can expose the buried--SH group, and this--SH group is essential for the catalytic activity of the enzyme. Two independent lines of evidence show that extensive polymerization of the enzyme caused by L-threonine and/or high concentrations of salt does not involve the formation of intermolecular disulfide bonds.
Subject(s)
Alcohol Oxidoreductases , Homoserine Dehydrogenase , Rhodospirillum rubrum/enzymology , Alcohol Oxidoreductases/metabolism , Amino Acids/analysis , Chloromercuribenzoates , Dithionitrobenzoic Acid , Homoserine Dehydrogenase/metabolism , Iodoacetates , Kinetics , Macromolecular Substances , Molecular WeightABSTRACT
Incubation of Rhodospirillum rubrum homoserine dehydrogenase (L-homoserine:NAD+ oxidoreductase, EC 1.1.1.3) with p-mercuribenzoate (PMB) in the presence of 0.2 M KCl and 2 mM L-threonine resulted in complete loss of enzyme activity. Upon removal of excess PMB, KCl, and L-threonine, a time-dependent recovery of enzyme activity was observed in 25 mM phosphate/I mM EDTA buffer, pH 7.5. Circular dichroism studies indicated that the transition from inactive to reactivated form of the enzyme was accompanied by a conformational change in the protein. Experiments with [14C]PMB revealed loss of enzyme-bound radioactivity during reactivation. Increase in ionic strength of the phosphate buffer and/or addition of L-threonine, leading to enzyme aggregation, decreased the rate of enzyme reactivation, aggregated enzyme that remained inactive retained [14C]PMB on the enzyme. Sulfhydryl titration of various forms of the enzyme suggested a preferential release of PMB from a sulfhydryl group essential to enzymic activity. We conclude that reactivation of the inactive enzyme is due to dissociation of PMB from an "active-site" sulfhydryl group and that changes in the protein structure influence the rate of dissociation of the enzyme-PMB complex.
