title: Purification and side chain selective chemical modifications of glutamate dehydrogenase from Bacillus subtilis natto.
abstract: Glutamate dehydrogenase (GDH) from Bacillus subtilis natto was purified to apparent homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, size exclusion chromatography, and hydroxyapatite (HA) affinity chromatography. The GDH was purified 34-fold, with a yield of 41 % of total activity and a specific activity of 34.29 U/mg proteins. The molecular weight (Mr) of was measured at 47 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 264 kDa with high-performance liquid chromatography (HPLC). The optimum pH and temperature for the deammoniation reaction were measured to be 7.5 and 30 °C, respectively. The active-site amino acid residues of GDH were investigated by chemical modification. The compounds 2,4,6-trinitrobenzenesulfonic acid (TNBS), phenylglyoxal (PG), and phenylmethanesulfonyl fluoride (PMSF) were used to modify lysine, arginine, and serine active site residues, respectively. After treatment with modifying reagents at concentrations of 1 mM, GDH activity fell to 10.7 % with TNBS, 83.3 % with PG, and 12.8 % with PMSF. However, with substrate protection, there was almost no loss in GDH activity following treatment with any modifying reagent. The kinetic parameters K m and V max were determined in each case. K m values for native GDH, 50 % TNBS-inactivated GDH, and 50 % PMSF-inactivated GDH were 0.037, 0.104, and 0.017 mM, respectively. V max values were 0.048, 0.022, and 0.031 mM/s, respectively. These results suggest that the active site contains one or more lysine residues that play a role in substrate binding and one or more serine residues that may maintain the enzyme conformation. However, arginine residues played less of a role in the activity of GDH.
authors.affiliation: School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Yinyun
authors.initials: Y
authors.lastname: Ni
authors.forename: Jiale
authors.initials: J
authors.lastname: Wang
authors.forename: Bingjun
authors.initials: B
authors.lastname: Qian
authors.forename: Guangyan
authors.initials: G
authors.lastname: Song
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Yao
authors.forename: Jian-hua
authors.initials: JH
authors.lastname: Zhang
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D011487:Protein Conformation; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D005969:Glutamate Dehydrogenase