Steady-state kinetics and inhibitor binding of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (tryptophan sensitive) from Escherichia coli

The tryptophan-inhibited 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [DAHPS-(Trp)] of Escherichia coli was analyzed with respect to steady-state kinetics and tryptophan binding. DAHPS(Trp) is one of three differentially regulated isoforms that catalyze the first step of aromatic biosynthesis, the condensation of phosphoenolpyruvate and erythrose-4-phosphate to form 3-deoxy-D-arabino-heptulosenate-7-phosphate. The DAHP synthase isozymes are metalloproteins, being activated in vitro by a variety of divalent metals. Both catalytic activity and substrate affinity are dependent on the species of activating metal ion. We report here kinetic and binding studies of metal homogeneous (Mn2+- activated) DAHPS(Trp). The homodimeric enzyme had an apparent k(cat) of 21 s(-1) and displayed sigmoidal kinetics with respect to both substrates. The S-0.5 was 35 mu M for erythrose-4-phosphate and 5.3 mu M for phosphoenolpyruvate. Equilibrium binding studies with radiolabeled tryptophan demonstrated two independent inhibitor binding sites per enzyme dimer, with K-d(Trp) of 1 mu M. L-Tryptophan binding decreased k(cat), increased affinity for both substrates, decreased positive homotropic cooperativity for both substrates and activated the enzyme at low concentrations of erythrose-4-phosphate. The results suggest an inhibition mechanism analogous to system C5 hyperbolic mixed-type inhibition with respect to erythrose-4-phosphate and partial noncompetitive inhibition with respect to phosphoenolpyruvate.