Differentially Regulated Isozymes of 3-Deoxy-d-arabino-Heptulosonate-7-Phosphate Synthase from Seedlings of Vigna radiata [L.] Wilczek

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  • RJRoy A. Jensen
    Roy A. Jensen
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Abstract
Two 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-4-phosphate (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 allosteric 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-d-arabino-heptulosonate-7-phosphate synthase and chorismate mutase are noted—one isozyme in each case being tightly regulated, the other being essentially unregulated.

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