3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase from Carrot Root (Daucus carota) Is a Hysteretic Enzyme

Abstract

Roots of carrots (Daucus carota) contain three activities of 3-deoxy-D-arabino-heptlsnte 7-phosphate (DAHP) synthase, the enzyme that catalyzes the first step of the shikimate pathway. The (10,19), Bacillus subtilis (13), Escherichia coli (17, 25), Salmonella typhimurium (11), Schizosaccharomyces pombe (2), and Brevibacterium flavum (29). The enzyme from cauliflower is the only plant DAHP synthase that has been substantially purified (14).Microorganisms employ a variety of mechanisms to control carbon flow through the shikimate pathway (8). While shikimate kinase appears to be the key allosteric control point of this metabolic route in B. subtilis (12) This paper shows that carrot roots contain three forms of DAHP synthase, enzymes I, II, and Ill; enzyme III is purified to electrophoretic homogeneity. This paper also presents a kinetic analysis that identifies enzyme III as hysteretic; hysteresis is affected by tryptophan, a feedback activator of the enzyme. MATERIALS AND METHODSReagents. PEP (4), erythrose-4-P (27), and DAHP (9) were synthesized and assayed as described previously (9, 25). All other reagents were obtained commercially at the highest purity available and were used without further purification.Enzyme Assays. DAHP synthase was assayed by either of two methods (25). In method A, the reaction mixture contained 0.75 Mmol of PEP, 0.3 Mmol of erythrose-4-P, 0.07 Mmol of L-tryptophan, 0.3 Mmol of MnCl2, and 100 ug of ovalbumin in a total volume of 0.1 ml of 50 mM bis-tris propane (pH 7.0). The reaction was initiated by adding 0.05 ml of appropriately diluted enzyme. The mixture was incubated for 20 min at 25C. The reaction was stopped with TCA. DAHP was determined after degradation with NaIO4 as the thiobarbiturate adduct. This endpoint assay was mainly used during the purification of the enzyme. In method B, 0.9 ml of appropriately diluted enzyme in 50 mm bis-tris propane (pH 7.0), containing 1 mM DTT and 150 Mm PEP were prewarmed to 25C. The reaction was started by addition of 100 Ml of 2.5 mm erythrose-4-P dissolved in the same buffer. The disappearance of PEP was continuously monitored by reading A232. This kinetic assay measures initial velocities and was used for most of the studies with the pure enzyme. One unit of DAHP synthase is defined as the amount of protein catalyzing the appearance of 1 Mmol of DAHP (method A) or the disappearance of 1 Mmol of PEP (method B) per min. Chorismate mutase was assayed as described (24).Protein Determination. Protein was determined by the method ofLowry et al. (15). Since DTT, bis-tris propane, and tryptophan interfere with this assay, protein was precipitated with 20% TCA and resuspended in 0.1 N NaOH, containing 0.2 M Na2CO3, prior to determination. During enzyme purification, protein in tryptophan containing buffers was determined by the method of Bradford (3) with lysozyme as the standard.Purification of DAHP Synthase Ill from Carrot Root (Dancus carota). All manipulations were carried out at 4C.Step . Fresh carrot roots were washed extensivel...

Redox Regulation of Arabidopsis 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase

“…Plant DAHP synthase was first purified to homogeneity from carrot (Daucus carota) roots and potato (Solanum tuberosum) tubers (Suzich et al, 1985;Pinto et al, 1986). Polyclonal antibodies specific for this protein were used to obtain cDNA encoding DAHP synthase (Dyer et al, 1990).…”

Glyphosate Induces 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase in Potato (Solanum tuberosum L.) Cells Grown in Suspension Culture

“…DAHP synthase activity also varies during the growth cycle of Daucus carota cells in suspension culture (29). One (25), and the pure D. carota enzyme shows hysteresis in response to tryptophan (30).…”

3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase from Potato Tuber (Solanum tuberosum L.)

“…No hysteresis is observed in the presence of 0.7 mm tryptophan. Like the DAHP synthases from carrot root (23) and cultured carrot cells (22), potato DAHP synthase is activated by tryptophan. In contrast, corn DAHP synthase is inhibited by tryptophan (6), the enzyme from pea leaves is inhibited by tyrosine (13), one bean DAHP synthase is inhibited by arogenate (16), and the enzymes from tea (17) and cauliflower (9) are not affected by the aromatic amino acids.…”

“…DAHP synthase activity has been demonstrated in several higher plants (2, 6, 9, 13-17, 22, 23); the enzyme has been extensively purified from Brassica oleracea (9), and has recently been obtained in homogeneous form from Daucus carota (23 …”

“…DAHP synthase activity has been demonstrated in several higher plants (2, 6, 9, 13-17, 22, 23); the enzyme has been extensively purified from Brassica oleracea (9), and has recently been obtained in homogeneous form from Daucus carota (23 MATERIALS AND METHODS Chemicals. P-Enolpyruvate (4) and erythrose-4-P (19) were synthesized and assayed as described (8,18).…”