Regulatory Enzymes of Aromatic Amino Acid Biosynthesis in Bacillus Subtilis: II. THE ENZYMOLOGY OF FEEDBACK INHIBITION OF 3-DEOXY-D-ARABINO-HEPTULOSONATE 7-PHOSPHATE SYNTHETASE

Abstract

Abstract Enzymological studies of the action of the inhibitors, chorismate and prephenate, upon purified preparations of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase in Bacillus subtilus are described for an allosteric system of feedback inhibition in aromatic amino acid biosynthesis. 1. The Ki values for chorismate and prephenate are 2.8 x 10-4 M and 4.0 x 10-5 M, respectively. 2. The following evidence is consistent with the absence of intersite interactions between substrate-binding sites: (a) the observation of first order kinetics in substrate saturation curves; (b) strictly noncompetitive inhibition; and (c) the determination of an interaction coefficient, n = 1.0, with the Hill system of coordinates. 3. Cooperative inhibitions were observed between inhibitor-binding sites. Plots of kinetic data take the form of sigmoid inhibition curves. The interaction coefficient, n', is 1.5 at inhibitor concentrations causing more than 35% inhibition of enzyme activity. Lower concentrations yield a value of 2.8 for n'. 4. Inhibition of DAHP synthetase by chorismate and prephenate is potentiated at low temperatures. This effect of temperature is correlated with the alteration of the overall dissociation constant, K. The cooperative interactions between substrate-binding sites are independent of temperature as judged by measurements of interaction coefficients at different temperatures. 5. Monomer-polymer protein interactions probably do not play a major role in the feedback inhibition of DAHP synthetase by intermediary metabolites in aromatic amino acid synthesis since the sedimentation pattern of the enzyme is unchanged in the presence of inhibitors. 6. DAHP synthetase is activated by p-chloromercuribenzoate and iodoacetate and these compounds potentiate the inhibition of the enzyme. In contrast, 2-mercaptoethanol inhibits DAHP synthetase and partially desensitizes the enzyme to inhibitor effects. 7. DAHP synthetase is partially desensitized with mercuric acetate. Heat treatment and exposure to urea have no effect upon inhibition of remaining enzyme activity. Total desensitization is never observed. Neither the feedback effectors nor the aromatic amino acids protected the enzyme from a number of inactivating agents.