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-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.
Akowski
J P
JP
Department of Biology, University of Virginia, Charlottesville, Virginia 22903-2477, USA.
Bauerle
R
R
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D008667:Metalloproteins; D013403:Sugar Phosphates; C026959:erythrose 4-phosphate; D010728:Phosphoenolpyruvate; D014364:Tryptophan; D010723:3-Deoxy-7-Phosphoheptulonate Synthase