Purification and properties of the 3‐deoxy‐d‐arabino‐heptulosonate‐7‐phosphate synthase (phenylalanine‐inhibitable) of Saccharomyces cerevisiae

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Abstract
The phenylalanine‐inhibitable 3‐deoxy‐d‐arabino‐heptulosonate‐7‐phosphate (dHplP) synthase from Saccharomyces cerevisiae has been purified to apparent homogeneity by a 1250‐fold enrichment of the enzyme activity present in wild‐type crude extracts, employing an overproducing strain. The estimated molecular mass of 42 kDa corresponds to the calculated molecular mass of 42.13 kDa deduced from the previously determined primary sequence. Gel filtration indicates that the active enzyme is a monomer. The enzyme is an Fe protein and is inactivated by EDTA in a reaction which is reversible by several bivalent metal ions. The Michaelis constant of the enzyme is 18 μM for phosphoenolpyruvate (P‐pyruvate) and 130 μM for erythrose 4‐phosphate (Ery4P) and the rate constant was calculated as 10 s−1. Inhibition by phenylalanine is competitive with respect to erythrose 4‐phosphate and non‐competitive to phosphoenolpyruvate, with a Ki of 10 μM.