Studies On The Conformational Changes Of Arginine Kinase By Site-directed Mutagenesis

Aginine kinase (AK) plays an important role in cellular energy metabolism in invertebrate,buffering cellular ATP levels by catalyzing the reversible phosphoryl transfer between ATP and arginine. Arginine kinase from Shrimp Metapenaeus ensis (M. ensis)(40 kDa)was taken a model to study conformational changes of protein. In this research, we replaced Cys271 with Ala or Ser, D62 with Glu or Gly and R193 with Lys or Gly by site-directed mutagenesis, respectively. All of the recombinant enzymes with 6xHis-tag were obtained from the E. coli strain Rosetta transformed with plasmids containing the genes for the wild-type and mutated AK, successfully purified by affinity chromatography, and confirmed to be highly purified by SDS-PAGEThe amino acid residues (Asp62 and Arg193) are responsible for the activity and structural stability of arginine kinase (AK). The amino acid residues Asp62 (D62) and Arg193 (R193) are strictly conserved in monomeric AKs and form an ion pair in the transition state analogue complex. The mutants of D62E and R193K retained almost 90% of the wild-type activity, whereas D62G and R193G had a pronounced loss in activity. A detailed comparison was made between the physic-chemical properties and conformational changes of wild-type AK and the mutants by means of fluorescence spectra. The results indicated that the conformation of all of the mutants had been changed and the stability in a urea solution was also reduced. We speculated that the hydrogen bond and electrostatic interactions formed between residues 62 and 193 play a key role in stabilizing the structure and mediating the synergism in substrate binding of arginine kinase.The role of Cys271 in conformational changes of AK. A detailed comparison of the catalytic activity and conformation was made between wild-type AK and the mutants by means of activity analysis, ultraviolet (UV) difference, fluorescence spectrum and size exclusion chromatography (SEC). The results indicated that the catalytic activity of the two mutants was almost gone. The substrates, arginine-ADP-Mg²⁺ could induce conformational changes, and additional NO3ˉcould induce further changes in both the native enzyme and the variants. We speculated that Cys271 might be located in the hinge region between the two domains of AK and cause enzyme conformational changes upon addition of substrate.