Purification and properties of glutamate synthase from Streptomyces lincolnensis

Glulamale synthase (EC 1.4.1. 14) was purified to homogeneity from 8 cell-free extract of Streptomyces lincolnensis by precipitation with streptomycin sulfate and ammonium sulfate, and column chromatography on DEAE cellulose, Sepharose 6B, DEAE-sephadex A-50, hydroxyapatite and Sephadex G-150. The enzyme activity is stabilized by addition of alpha-ketoglutarate, PMSF, EDTA, beta-mercaptoethanol and glycerol. The native enzyme has a molecular weight of 138 000 and is composed of two nonidentical subunits with molecular weights of 81 000 and 57 000. Spectroscopic examination of the enzyme gave absorption maximum at 280 and none at 380 and 440 nm, indicating the absence of iron and flavin. The enzyme shows optimum activity at pH 7.2 and 30 degrees C. Km values for alpha-ketoglutarate, L-glutamine and NADH were 417, 435, and 52.1 micromol/L, respectively. When NADPH was substituted lor NADH as reductant, there was approximately 13% of the control activity. The activity of this glutamate synthase is inhibited by its products (i.e. glutamate and NAD), several metal ions, amino acids and tricarboxylic acid cycle intermediates.