ABSTRACIrTwo isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) designated DS-Mn and DS-Co were separated from seedlings of Vigna radiata [L.] Wilczek by DEAE-cellulose column chromatography. DS-Mn was activated 2.6-fold by 0.4 millimolar manganese, had an activity optimum of 7.0, and was substrate inhibited by erythrose-4phosphate (E4P) concentrations in excess of 0.5 millimolar. In contrast, DS-Co had an activity optimum at pH 8.8, required E4P concentrations of at least 4 millimolar to approach saturation, and exhibited an absolute requirement for divalent cation (cobalt being the best). Regulatory properties of the two isozymes differed dramatically. The activity of DS-Mn was activated by chorismate (noncompetitively against E4P and competitively against phosphoenolpyruvate), and was inhibited by prephenate and L-arogenate (competitively against E4P and noncompetitively against phosphoenolpyruvate in both cases). Under standard assay conditions, L-arogenate inhibited the activity of DS-Mn 50% at a concentration of 155 micromolar and was at least 3 times more potent than prephenate on a molar basis. Weak inhibition of DS-Mn by L-tryptophan was also observed, the magnitude of inhibition increasing with decreasing pH. The pattern of allosteric control found for DS-Mn is consistent with the operation of a control mechanism of sequential feedback inhibition governing overall pathway flux. DS-Co was not subject to aliosteric control by any of the molecules affecting DS-Mn. However, DS-Co was inhibited by caffeate (3,4-dihydroxycinnamate), noncompetitively with respect to either substrate. The striking parallels between the isozyme pairs of 3-deoxy-n.arabino-heptulosonate-7-phosphate synthase and chorismate mutase are noted-one isozyme in each case being tightly regulated, the other being essentially unregulated. 17, 1984, at Davis, CA. 2Abbreviations: DAHP, 3-deoxy-i-arabino-heptulosonate-7-phosphate; PEP, phosphoenolpyruvate; E4P, erythrose-4phosphate. marins, indole derivatives, and other phenolic compounds (12).In mung bean we showed (25) that total DAHP synthase activity was distributed between two separable and quite distinctive isozymes. One isozyme (DS-Co) had an absolute requirement for divalent cation (cobalt being preferred). The second isozyme (DS-Mn) was stimulated by, but did not require, manganese. Although Nicotiana silvestris is not closely related to V. radiata, a pair of isozymes similar to the V. radiata isozymes were noted in N. silvestris (7,8). This latter uniformity ofisozyme makeup is not reflected, however, by descriptions in the literature of DAHP synthase from corn (14), cauliflower (15), pea (20,22,23), tea (26), and carrot (28). Since we have found that assay conditions that favor the optimal detection of one isozyme are inadequate for detection of the second isozyme and vice versa, it is quite possible that this two-isozyme system may have escaped recognition in some other studies. For example, although a single enzyme that appears similar to DS-Mn wa...

The enzymes of the 4-hydroxyphenylpyruvate (prephenate dehydrogen-ase and 4-hydroxyphenylpyruvate aminotransferase) and pretyrosine (pre-phenate aminotransferase and pretyrosine dehydrogenase) pathways of L-tyrosine biosynthesis were partially purifled from mung bean (Vigna radiata IL.1 Wilczek) seedlings. NADP-dependent prephenate dehydrogenase and pretyrosine dehydrogenase activities coeluted from ion exchange, adsorp-tion, and gel-ftration columns, suggesting that a single protein (52,000 daltons) catalyzes both reactions. The ratio of the activities of partially purified prephenate to pretyrosine dehydrogenase was constant during al purification steps as weU as after partial inactivation caused by p-hydrox-ymercuribenzoic acid or heat. The activity of prephenate dehydrogenase, but not of pretyrosine dehydrogenase, was inhibited by L-tyrosine at nonsaturating levels of substrate. The K. values for prephenate and pretyrosine were similar, but the specific activity with prephenate was 2.9 times greater than with pretyrosine. Two peaks of aromatic aminotransferase activity utilizing L-glutamate or L-aspartate as amino donors and 4-hydroxyphenylpyruvate, phenylpyr-uvate, and/or prephenate as keto acid substrates were eluted from DEAE-cellulose. Of the three keto acid substrates, 4-hydroxyphenylpyruvate was preferentially utilized by 4-hydroxyphenylpyruvate aminotransferase whereas prephenate was best utilized by prephenate aminotransferase. The identity of a product of prephenate aminotransferase as pretyrosine following reaction with prephenate was established by thin layer chromatography of the dansyl-derivative. The shikimate pathway of aromatic amino acid biosynthesis begins with the condensation of erythrose-4-P and phosphoenol-pyruvate and culminates in the formation of L-tryptophan, L-tyrosine, and L-phenylalanine. Although many of the pathway reactions have been studied in higher plants (for reviews, see 2 and 23), the enzymology of the distal branches of the shikimate pathway, leading from prephenate to L-tyrosine and L-phenylal-anine, has neither been fully investigated nor evaluated for the presence of biosynthetic routes to L-phenylalanine or L-tyrosine that proceed via pretyrosine. Previous studies showed the following. Labeled tyrosine or phenylalanine was formed when young plant shoots were fed radioactive 4-hydroxyphenylpyruvate or phenylpyruvate (12). Cell-free extracts of plants catalyzed the following enzymic conversions: 4-hydroxyphenylpyruvate to tyrosine; phenylpyruvate to ' This study represents part of a thesis presented by Judith L. Rubin to the Graduate School of the State University of New York at Binghamton in partial fulfillment of the requirements for the M.A. degree. The study was supported in part by Department of Energy Contract EP-78-S-02-4967. phenylalanine; and prephenate to 4-hydroxyphenylpyruvate, ty-rosine, and phenylalanine (13). Prephenate dehydrogenase and an aromatic aminotransferase (utilizing tyrosine to form 4-hydroxy-phenylpyruvate) were partially purified and studie...

The effects of 1 milliolar glyphosate (N-Iphosphonomethyllglycine) upon the activities ofenzymes of aromatic amino acid biosynthesis, partially purified by ion-exchange chromatography from mung bean seedings (Vigna radiata IL.I Wilczek), were examined. Multiple isozyme species of shikimate dehydrogenase, chorismate mutase, and aromatic aminotransferase were separated, and these were all insensitive to inhibition by glyphosate. [271, and tobacco [unpublished data]) are now coming to light. Second, the particular details of reversal of glyphosate inhibition by aromatic acids are variable from organism to organism. In E. coli, all three aromatic amino acids are required for complete reversal (25). In Rhizobium japonicum, the combination of phenylalanine plus tyrosine reversed glyphosate inhibition (18) in contrast to Lemna gibba where phenylalanine alone was adequate for reversal (18). In Euglena gracilis, aromatic amino acids only partially reverse inhibition (40%o) unless the minor pathway products: 4-aminobenzoate, 4-hydroxybenzoate, 2,3-dihydroxybenzoate, and 3,4-dihydroxybenzaldehyde are also present (5).Where partial reversal of inhibition is obtained with either Lphenylalanine alone or L-tyrosine alone, the greatest reversal is usually accomplished by L-phenylalanine. For example, the antagonism of inhibition by L-phenylalanine and L-tyrosine was 41% and 12% in Escherichia coli, and 27% and 3% in Chlamydomonas reinhardii (15). Qualitatively, it was found that L-phenylalanine was a better partial antagonist of glyphosate than L-tyrosine in suspension cultures of both carrot and soybean (15)

Treatment of isogenic suspension-cultured cells of Nicotiana silvestrisSpeg. et Comes with glyphosate (N-phosphonomethyljglycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-D-arabiaoheptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ionexchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pK. values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO-CH2NH2+CH2PO32-, and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (K = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (K,' = 183 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an lenzyme:shikimate-3-PJ complex and ultimately forms the dead-end complex of lenzyme:shikimate-3-P:glyphosatel.Glyphosate (N-[phosphonomethyl]glycine) is a broad-spectrum herbicide which is also a potent growth inhibitor ofbacteria and algae (14). Although glyphosate may ultimately perturb a variety of biochemical processes including protein synthesis, nucleic acid synthesis, photosynthesis and respiration (16), the early conjecture (18)

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