Regulatory Enzymes of Aromatic Amino Acid Biosynthesis in Bacillus subtilis: I. PURIFICATION AND PROPERTIES OF 3-DEOXY-D-ARABINO-HEPTULOSONATE 7-PHOSPHATE SYNTHETASE

Abstract

Abstract 1. The 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase of Bacillus subtilis has been purified 77-fold (more than 500-fold over the activity in crude extracts of wild-type strains grown in a minimal salts medium). 2. The following points support the thesis that DAHP synthetase is a single enzyme: (a) The sensitivity of the enzyme to two inhibitors is constant throughout a 77-fold purification. (b) Prephenate and chorismate independently inhibit the enzyme activity 80% at saturating inhibitor concentrations. (c) Fractionation procedures yield nonseparable DAHP synthetase activities which are similar in properties such as storage stability, thermostability, inactivation by urea or low pH, and in experimental values of kinetic constants of enzyme velocity. (d) The logic for one DAHP synthetase as the initial metabolic control point for pathway-wide regulation is consistent with efficient control by the feedback inhibitors, chorismate and prephenate. (e) Auxotrophs which are deficient for DAHP synthetase occur as single step revertible mutations. 3. Modifications in the assay for DAHP synthetase activity in B. subtilis are described. 4. Partially purified preparations of enzyme have a pH optimum of 6.5, a pH stability optimum of 6.9, and a temperature optimum of 55°. An activation energy of 9,100 cal per mole was calculated. The enzyme is activated 2-fold by low concentrations of p-chloromercuribenzoate and iodoacetate and inhibited by concentrations of 2-mercaptoethanol exceeding 10-5 M. Molecular weights of 134,000 and 129,000 were determined by gel filtration and sucrose density gradient techniques, respectively.