Studies On The Structure And Characterization Of Arginine Kinase

Aginine kinase (AK) plays an important role in cellular energy metabolism in invertebrate. Arginine kinase from Shrimp Metapenaeus ensis (M. ensis)(40 kDa)was taken a model to study structure and characterization of protein.The encoding AK gene from Shrimp Metapenaeus ensis (M. ensis) was cloned in prokaryotic expression plasmid pET-28a, and it was then expressed in Escherichia coil in dissoluble form.The recombinant protein was purified by following three chromatography steps in turn:CM-Cellulose cation-exchange, Sephacryl S-100 HR gel filtrate and DEAE-Sepharose anion-exchange. The purified AK's apparent Km was 2.33±0.1 mM and 1.59±0.2 mM for ATP and L-arginine, respectively, while its optimum pH and temperature was 8.5 and 30℃in the process of forward reaction, respectively.With the method of site-derected mutagenesis, Tyr68 is replaced by Trp and Ala respectively, and were expressed in E.coli. Rosseta. The two mutant proteins were carried out separation and purification by using His-Tag nickel affinity chromatography column. The effects of site-directed mutagenesis at residue Tyr68 on AK conformation and catalytic activity were investigated by the means of activity analysis, fluorescence spectrum, size exclusion chromatography (SEC) and trypsin digestion. The results revealed that the catalytic activities of two mutants were both completely lost, and accompanied by a series of conformational changes,including apparent molecular weight increase, intrinsic fluorescence emission peak significant red-shift, protein hydrophobic surface enlargement and more susceptible to trypsin digestion. These data suggested that the residue Tyr68 played an essential role in the maintenance of conformational stability and catalytic function of arginine kinase.