Corn leaf glutamate synthase: Purification and properties of the enzyme

Abstract

An assay for ferredoxin-glutamate synthase is introduced that uses an anion exchange resin to isolate the glutamate formed and subsequent determination with the ninhydrin procedure. The enzyme was purified 200-fold from corn leaves by ammonium sulfate fractionation and chromatography on DEAE-cellulose, DEAE-Sephacel and ferredoxin- Sepharose. The purified enzyme had a specific activity of 14 μmoles glutamate formed min⁻¹mg⁻¹ protein. The enzyme has a molecular weight of 160,000. The pH optimum for catalytic activity is 6.9. The isoelectric point is at pH 4.2. The apparent Km values of the enzyme for L-glutamine, 2-oxoglutarate and ferredoxin are 1,100, 240 and 1.7 μM. The enzyme has a high specificity toward these substrates with a stoichiometry between glutamate formation and glutamine consumption. Sulfhydryl reagents, bathophenanthroline, phthalein acids and azaserine produced strong inhibition of the enzyme activity.