title: Mutation of residue arginine 330 of arginine kinase results in the generation of the oxidized form more susceptible.
abstract: Arginine kinase (AK), a crucial enzyme for the energy metabolism of invertebrates, catalyzes the reversible phosphorylation of arginine by Mg(2+)ATP to form phosphoarginine and Mg(2+)ADP. Arginine 330 (R330), not involved in the catalysis of phosphoryl transfer, is a residue highly conserved in the phosphagen kinase family. In order to investigate the role of R330 in AK, it was replaced by lysine (R330K). Non-reduced SDS-PAGE analysis suggested that wild type AK (Wt-AK) and R330K existed in two forms, the reduced form (R-AK or R-R330K) and the oxidized form (O-AK or O-R330K), whereas O-R330K was more susceptible to generate than O-AK. Subsequently, an intramolecular disulfide bond in O-R330K was demonstrated to be formed between Cys201 and Cys271 by site-directed mutagenesis. Biochemical analysis revealed that conformational changes of R330K were concomitant with the sharp decline of catalytic activity. These results were further confirmed by structure modeling of AK and R330K. Therefore, it can be concluded that R330 residue plays an important role in the structural stability and activity of AK.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Wei-Dong
authors.initials: WD
authors.lastname: Wang
authors.forename: Jin-Song
authors.initials: JS
authors.lastname: Wang
authors.forename: Yu-Lin
authors.initials: YL
authors.lastname: Shi
authors.forename: Xin-Chao
authors.initials: XC
authors.lastname: Zhang
authors.forename: Ji-Cheng
authors.initials: JC
authors.lastname: Pan
authors.forename: Guo-Lin
authors.initials: GL
authors.lastname: Zou
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics*; D001122:Arginine Kinase / Q000737:chemistry / Q000235:genetics*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003445:Crustacea / Q000201:enzymology*; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
chemical_list: D004220:Disulfides; D050505:Mutant Proteins; D001120:Arginine; D001122:Arginine Kinase; D003545:Cysteine