3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase of Streptomyces aureofaciens Tü 24. I. Partial purification and properties.

Abstract

Abstract 1. 1. 3-Deoxy- d -arabino- heptulosonate-7-phosphate synthase ( 7-phospho-2-oxo-3-deoxy- d -arabino- heptonate d -erythrose-4-phosphate-lyase (pyruvate phosphorylating), EC 4.1.2.15, DAHP synthase), the first enzyme of the aromatic amino acid pathway, has been purified 170 fold from Streptomyces aureofaciens Tu 24. DAHP synthase is a single enzyme with a molecular weight of 97 000. 2. 2. The enzyme has a broad pH stability optimum between pH 6 and 8. The catalytic activity has a pH optimum of 7. EDTA causes a partial inactivation of the enzyme, which can be reversed by Co 2+ . The activation energy of the enzymatic reaction was estimated to be 16 500 cal/mole. Heat inactivation occurs above 50°. Phosphoenolpyruvate protects the enzyme against heat inactivation, whereas the presence of erythrose 4-phosphate increases the rate of inactivation. 3. 3. The initial velocity data follow regular Michaelis-Menten kinetics without any detectable kinetic evidence for subunit interaction. On the basis of kinetic experiments the mechanism of enzyme action is ping-pong, and the first substrate is phosphoenolpyruvate. The absolute Michaelis constants of the enzyme have been determined to be 3·10 −4 M for phosphoenolpyruvate and 1.6·10 −4 M for erythrose 4-phosphate. 4. 4. Quantitative determination of phosphoenolpyruvate and erythrose 4-phosphate by the use of a DAHP synthase preparation of Streptomyces aureofaciens is described. The method shows a deviation of ±0.7% from the theoretical values.