.GAMMA.-Glutamylcysteine synthetase from Proteus mirabilis.

The distribution of γ-glutamylcysteine synthetase (L-glutamate: L-cysteine γ-ligase, EC 6.3.2.2) was investigated in bacteria, and the enzyme was purified from Proteus mirabilis approximately 9, 000-fold with an over-all yield of 10%. The purification procedure included ammonium sulfate fractionation, protamine treatment, DEAE-cellulose and- hydroxylapatite column chromatographies and Sephadex gel filtrations. The purified enzyme was homogeneous by the criteria of ultracentrifugation. It showed multiple bands on disc-polyacrylamide gel electrophoresis and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band with a molecular weight of 62, 000 was obtained on SDS-polyacrylamide gel electrophoresis after cross-linking of the enzyme with dimethylsuberimidate. The molecular weight was determined from the sedimentation and diffusion coefficients to be 64, 000 and by Sephadex G-150 gel nitration to be 62, 000. The purified enzyme catalyzed the stoichiometric formation of γ-glutamylcysteine and the reaction showed a sigmoidal dependence upon L-cysteine concentration. The enzyme also catalyzed γ-glutamyl amino acid formation from L-α-aminobutyrate, L-homoserine, glycine, L-serine, DL-norvaline or DL-homocysteine, but at lower rates than from L-cysteine. The γ-glutamyl-α-aminobutyrate formation by the enzyme did not show a sigmoidal but a hyperbolic dependence upon L-α-aminobutyrate concentration.