Control of isoleucine, valine, and leucine biosynthesis. I. Multivalent repression.

Repression of the formation of biosynthetic enzymes by the ultimate end product of their action has been shown to be an important aspect of metabolic regulation in microorganisms.1 In Escherichia coli and Salmonella typhimuwium each of the last four steps in the synthesis of valine and isoleucine is catalyzed by the same enzymes.2 Repression of these enzymes presents a unique situation since two ultimate end products are formed by their action. In addition, the initial reaction leading to leucine biosynthesis involves an intermediate in the valine pathway.3 In this report we would like to describe experiments which indicate that valine, isoleucine, and leucine are required for the repression of the enzymes necessary for isoleucine and valine biosynthesis. In contrast to these results, the enzymes of the leucine pathway appear to be repressed only by leucine. Material7s and Method&.-Organisms: The following derivatives of S. typhimnurilum strain LT-2 were used: leit 130 ilva 224 which lacks enzymes 3 and 7 (Fig. 1) and therefore requires isoleucine, valine, and leucine; leu 124 which lacks the initial enzyme in leucine biosynthesis (Fig. 1, enzyme 6); and ilva A-8 which is an isoleucine-valine auxotroph deficient in enzyme 3 (Fig. l) anld which was kindly supplied by Dr. M. Demerec. A mutant derived from E. coli strain W, M4862-H5, Which has a partial block in enzyme 3 and lacks enzyme 5 (Fig. 1), was employed. It has an absolute requirement for isoleucine and valine and a partial requirement for leucine. Chemicals: a-Hydroxy-fl-carboxyisocaproate was isolated from culture filtrates of Neurospora crassa using the method of Gross et al.4 a,0-I)ihydroxyisovalerate was prepared according to the procedure of Sjolander et al.5 Other compounds were of reagent grade. Enzyme methods: For this study the following enzymes were examined: (1) L-threonine dealminase (Fig. 1, enzyme 1) Which is involved only in the formation of isoleucine; (2) dihydroxyacid dehydrase (Fig. 1, enzyme 4) which is necessary for the biosynthesis of isoleucine and valine and (3) a-hydroxy-_#-carboxyisocaproate decarboxylase (Fig. 1. enzyme 8) which is required solely for the synthesis of leucine. The procedure for growth of the organisms, preparation of extracts, and measurement of threonine deaminase activity has been described previously.' The chemostat experiments followed the usual method.7 Dihydroxyacid dehydrase activity was measured according to the procedure of Meyers and Adelberg8 except that in the assay a,,B-dihydroxyisovalerate was increased to 20 5mole per ml, the buffer was pH 8.0, and MgCl2 was employed in place of MgSO4. aHydroxy-3l-carboxyisocaproate decarboxylase activity was determined by measuring the amount of a-ketoisocaproate formed by the indirect ketoacid procedure of Friedemann and Haugen.9 The assay mixture contained per 2 ml: Tris-HCl buffer, pH 8.0, 300 ,mole; MnCI1, 1.0 umole; KCl, 100 /fmole; DPN, 2 ,umole, a-hydroxy-3-carboxyisocaproic acid, 1.0 Amole, and crude bacterial extract. The mixture was incubated at 370C for 10 min after which 3 ml of phenylhydrazine reagent was added to stop the reaction. The specific activity for all the enzymes examined is expressed as the Mmoles of keto acid formed per mg protein per hr. Results.-The results in Table 1 reveal that an excess of isoleucine, valine, and leucine was needed to repress L-threonine deaminase and dihydroxyacid dehydrase. When any one of these amino acids was added in limiting amounts, in the presence of an excess of the other two compounds, a 5to 10-fold increase was noted in enzyme activity. In contrast, the activity of the decarboxylase, which is specific for leucine formation, increased only when the cells were grown in the presence of