PURIFICATION AND PROPERTIES OF 3-DEOXY-D-ARABINO-HEPTULOSONATE-7-PHOSPHATE SYNTHASE(PHENYLALANINE SENSITIVE) OF ESCHERICHIA COLI K12 .I. PURIFICATION OF ENZYME AND SOME OF ITS CATALYTIC PROPERTIES

1. 1. The phenylalanine-sensitive allosteric first enzyme (isoenzyme 1a) of the aromatic amino acid biosynthetic pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (7-phospho-2-oxo-3-deoxy-d-arabino-heptonated-erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC 4.1.2.15) has been purified 160-fold from Escherichia coli K12. The molecular weight of the enzyme is 160 000. 2. 2. The enzyme has a broad pH optimum between pH 6 and 8. The initial velocity data obtained 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 phosphoenol-pyruvate. The absolute Michaelis constant of the enzyme for both phosphoenol-pyruvate and erythrose 4-phosphate is 1.0 mM. Co2+, at a concentration of 1 mM, increases the enzyme activity about 2-fold. Among other ions tested, Mn2+ slightly activates the enzyme, while other heavy metal ions, such as Cu2+ and Zn2+, are inhibitory. 3. 3. Both elevated and low temperatures reversibly inactivate the enzyme. Phosphoenolpyruvate protects the enzyme against the inactivating effect of heat. The Ks of the enzyme for phosphoenolpyruvate depends on the temperature and decreases with decreasing temperature. © 1969.