3-deoxy-D- arabino-heptulosonate-7-phosphate synthetase of Mycobacterium phlei: Partial purification and feedback inhibition

Abstract

Summary 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase (E.C.4.1.2.15), the first enzyme of the aromatic amino acid biosynthetic pathway, has been studied in a Mycobacterium phlei strain, cultivated in a minimal salt medium, either as surface grown cells (conditions I) or as shaken medium cultures (conditions II). Conditions I were retained to obtain a 14-fold purified fraction subsequently used for the study of the enzymatic activity. An optimum pH at about 6.6 is observed. Co++ is essential for activity and Zn++ is a strong inhibitor. The Km value is 3.5 mM for phosphoenolpyruvate and 0.45 mM for erythrose-4-phosphate. Under conditions I, each aromatic amino acid inhibits weakly the activity, while under conditions II a higher inhibition was observed with L-tyrosine and L-phenylalanine. Under both conditions, a synergistic inhibitory effect is observed with the three aromatic amino acids together, with a maximum effect around 0.1 mM for each amino acid under conditions I and 0.01 mM under conditions II. A similar synergistic effect is also obtained with the two tryptophan containing pairs, and the inhibition attained is nearly equal to the maximal inhibition observed when the three aromatic amino acids are present (about 75 p. cent of total activity). L-phenylalanine plus L-tyrosine have only a slight synergistic effect, highly enhanced by the addition of L-tryptophan. From the synergistic inhibition characteristics of the enzyme joined to the fact that subsequent fractionation procedures failed to separate different DAHP-synthetase activities, it is concluded that M. phlei contains a single DAHP-synthetase, with a molecular weight of about 130,000, as determined by gel filtration and sucrose density gradient.