The kinetics of a purified form of 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (tryptophan) from Neurospora crassa.
1. A method is described for the purification of a form of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (tryptophan) that probably differs from that of the native enzyme. 2. The kinetics of the reaction catalysed by 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (tryptophan) shows that the reaction proceeds via a ping-pong bi-bi mechanism, with activation by phosphoenolpyruvate (P-Prv), the first substrate, and inhibition by erythrose 4-phosphate (Ery-P) the second substrate. At low substrate concentrations, KP-Prv is 0.1 mM and KEry-P is 0.13 mM. 3. The substrates phosphoenolpyruvate and erythrose 4-phosphate and the product inorganic phosphate can protect the purified enzyme against heat denaturation, whereas the inhibitor, tryptophan, has no effect, although it binds to the enzyme in the absence of other ligands. 4. Product inhibition by inorganic phosphate is linear non-competitive with respect to phosphoenolpyruvate (Ki, slope = 22 mM and Ki, intercept = 54 mM) and substrate-linear competitive with respect to erythrose 4-phosphate (Ki, slope = 25 mM). 5. The enzyme has an activity optimum at pH 7.3 and a tryptophan inhibition optimum at pH 6.4, Trp 0.5 is 4 microM. Inhibition by tryptophan is non-competitive with respect to phosphoenolpyrovate and substrate-parabolic competitive with respect to erythrose 4-phosphate. 6. The role of the enzyme in metabolic regulation is discussed.
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000302:isolation & purification / Q000378:metabolism*; D000446:Aldehyde-Lyases / Q000378:metabolism*; D004355:Drug Stability; D007700:Kinetics; D008024:Ligands; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D014364:Tryptophan
D008024:Ligands; D014364:Tryptophan; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D000446:Aldehyde-Lyases