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-heptulosonate-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 kcat of 21 s-1 and displayed sigmoidal kinetics with respect to both substrates. The S0.5 was 35 microM for erythrose-4-phosphate and 5.3 microM for phosphoenolpyruvate. Equilibrium binding studies with radiolabeled tryptophan demonstrated two independent inhibitor binding sites per enzyme dimer, with KdTrp of 1 microM. L-Tryptophan binding decreased kcat, 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.