Effects of K+ on the catalytic and regulatory properties of homoserine dehydrogenase of Pseudomonas fluorescens

Abstract

Partially purified homoserine dehydrogenase (-homoserine; NADP+ oxidoreductase, EC 1.1.1.3) isolated from Pseudomonas fluorescens requires K+ for its stability. At 1 mM K+, the apparent first-order rate constant for enzyme inactivation at 25[deg] was 25-fold higher than that observed at 20 mM K+. A dissociation constant, KD, of 3 mM of the ion-enzyme complex was calculated.Following inactivation of the enzyme by depletion of K+, the activity could be regenerated to a large extent by incubation with KCl and NADP+; no other cation was nearly as effective as K+.Binding of K+ on the enzyme molecule also plays an important part in the regulation of enzyme activity by the allosteric modifier -threonine. At 1 mM K+, the catalytic activity was strongly inhibited by 10 mM -threonine, whereas, at 10 mM K+ concentration in the assay mixture, -threonine inhibition was almost completely abolished; threonine and K+ were kinetically competitive. It is proposed that K+ can induce specific conformational changes in the protein molecule that are either sensitive or insensitive to feedback inhibition control; in addition, the cation is necessary for the maintenance of the protein structure to ensure catalytic activity.