Regulation of Aromatic Amino Acid Biosynthesis in Higher Plants: II. 3-DEOXY-ARABINO-HEPTULOSONIC ACID 7-PHOSPHATE SYNTHETASE FROM CAULIFLOWER

Abstract

Abstract 3-Deoxy-arabino-heptulosonic acid 7-phosphate synthetase, which catalyzes the condensation of P-enolpyruvate and erythrose-4-P to yield 3-deoxy-arabino-heptulosonic acid 7-phosphate has been purified 1000-fold from cauliflower florets. The enzyme exhibited an activity optimum around pH 6.6 and a stability optimum around pH 7.7. Optimum enzyme stability was achieved in a buffer of pH 7.7, containing a reducing agent, MnCl2, and a polyol compound such as glycerol or 1,3-propanediol. Loss of synthetase activity occurred rapidly in preparations with low protein concentration, but could be prevented by addition of bovine serum albumin. The reaction catalyzed by the enzyme was linear both with time and protein concentration. The synthetase exhibited high specificity for the substrates erythrose-4-P and P-enolpyruvate. An activation energy of 17,700 cal per mole was calculated for the enzymatic reaction. Strong inhibition by sulfhydryl reagents indicated that the synthetase contains a —SH group which is essential for activity. Inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) was reversed by dithiothreitol. Inhibition of activity by a number of metal chelators indicates that the enzyme contains a metal ion important for activity. Of eight cations tested, Mn2+ was most active in reversing EDTA inhibition; Ni2+, Co2+, Zn2+, and Cu2+ inhibited enzyme activity. Although the addition of Mn2+ was effective in stabilizing the enzyme, its addition was not necessary for demonstrating enzyme activity. Double reciprocal plots of velocity versus concentration of P-enolpyruvate were biphasic. S0.5 values for erythrose-4-P of 0.24 and 0.14 mm were calculated for high and low concentrations of P-enolpyruvate, respectively. The corresponding S0.5 values for P-enolpyruvate were 0.30 and 0.025 mm at high and low concentrations of P-enolpyruvate, respectively. Inhibition by erythrose-4-P occurred at concentrations greater than 1 mm but the inhibition was reversible by P-enolpyruvate. Gel chromatography of partially purified enzyme preparations gave evidence for the existence of several forms of the enzyme with different molecular weights. The enzyme was not inhibited by chorismate, phenylalanine, tyrosine, or tryptophan.