Homoserine dehydrogenase: spontaneous reactivation by dissociation of p-mercuribenzoate from an inactive enzyme--p-mercuribenzoate complex.
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.
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D002942:Circular Dichroism; D004789:Enzyme Activation; D006715:Homoserine Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008626:Mercuribenzoates / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000378:metabolism
D008024:Ligands; D008626:Mercuribenzoates; D013438:Sulfhydryl Compounds; D000429:Alcohol Oxidoreductases; D006715:Homoserine Dehydrogenase
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