Studies On The Active Site Of Arginine Kinase From Sea Cucumber Stichopus Japonicus By Site Mutagenesis

This paper studies on the arginine kinase from sea cucumber Stichopus japonicus. This enzyme is a phosphagen kinase, it is homodimer with molecular weight of 42 kD per subunit, and its isoelectric point is 8.05.Three mutations: Cys274Ala (C274A), Arg283Gly (R283G) and His287Ala (H287A) of sea cucumber arginine kinase were obtained in this paper by site mutagenesis. It was expressed in E.coli. The bulk of expressed protein resided in insoluble inclusion bodies. The catalytic activity and some structural feature have been analyzed. The secondary structure has almost no change. But the catalytic activity was lost completely. The result of ANS binding fluorescence indicated that the hydrophobic surface of the mutant R283G and H287A were all decreased compared to the wild-type AK, while hydrophobic surface of the mutant C274A has almost no change. Result of size exclusion chromatography (SEC) indicated that the molecular structure of the mutant C274A was a little tighter than wild type AK, while those of the mutant R283G and H287A were a little looser than wild type AK. Result of Native-PAGE electrophoresis indicated that the positive electrical charge of the mutant C274A was increased, while the negative electrical charge of the mutant R283G and H287A were all increased. Taken together, we conclude that all of these three residues play important roles in either structural or functional keeping.It has been reported that Cys283 in the rabbit muscle creatine kinase is the active site, and this residue is a highly conservative site among creatine kinases and arginine kinases, Cys274 in sea cucumber arginine kinase for example. The modified Cys residue with OPTA proved the only one Cys residue is necessary for the activity of sea cucumber arginine kinase. In the crystal structure of horseshoe crab, some one has pointed out Cys271 in AK sequence has a certain function in the linking of the arginine and the enzyme. We can know that Cys274 in the sea cucumber AK sequence has the similar function with Cys271 in the horseshoe crab, so it might be the active center of the enzyme.