Dissociation‐produced loss of regulatory control of homoserine dehydrogenase of rhodospirillum rubrum

HDH *** ofRhodospirillum rubrum is subject to feedback control by several amino acid end products of the pathway in which it functions [ 11. Evidence for the reversible aggregation of the enzyme under the influence of ligands has come from density gradient and gel fdtration experiments [2]. In this paper we present kinetic data which indicate that the enzyme undergoes a reversible dissociation to a form which is insensitive to feedback inhibition by threonine, as well as to inhibition by the threonine analog &hydroxynorvaline, and which is also insensitive to activation by methionine and isoleucine. The equilibrium is shifted toward the sensitive form by preincubation with these ligands, as well as certain cations and anions, and toward the modulator-insensitive form by homoserine and by serine, an inhibitory substrate analog. with the elimination of contaminating aspartic acid by Dowex AG 5OW-X8 (H+) chromatography. Buffer A contained 30 mM potassium phosphate, 1 mM K,EDTA and 0.1 M KCl, pH 7.2, p = 175 mM. The standard enzyme assay was in 3.1 ml of buffer A containing 15 PM TPNH and 58 PM L-ASA at 23” in a Cary model 11 spectrophotometer with a 0 to 0.1 absorbancy slide wire and a 1 cm light path. One unit of enzyme is defined as the amount catalyzing the oxidation of 1 I.tmole of TPNH under these conditions. Lthreonine, when added to the assay cuvette, was at a concentration of 0.48 mM. Protein was determined by the method of Lowry et al. [4] _ R. rubrum (a gift of Dr. H.Gest) was grown photosynthetically under anaerobic conditions in a synthetic malate medium [ 11. The cells were harvested after 48-60 hr of growth. The enzyme was extracted by sonication and purified about 16-fold before use by ammonium sulfate fractionation and was in buffer A.