Studies On The Structure And Function Of Arginine Kinase Based On Site-directed Mutagenesis

An greasyback shrimp （Metapenaeus ensis） Arginine Kinase is studied in thispaper. It is a phosphagen kinase that onsists of40KD single subunit and mainlyexisits in cells of invertebrates, which plays an important buffer role in its energymetabolismIn this paper the eight mutants of Chinese shrimp arginine kinase has acquired.through inverse PCR to site-directed mutagenesis of the wild-type AK gene:Asp226Glu （D226E）, Asp226Asn （D226N）, Asp226Ala （D226A）, Tyr89Phe （Y89F）,Tyr89Asp （Y89D）, Arg330Lys （R330K）, Arg330Lue （R330L） andTyr89Phe/Arg330Lue （Y89F/R330L）, and successfully soluble expressed in E. coliRosetta, because eight mutant and wild-type AK has6His-tag, high electrophoreticpurity of the target protein can be obtained by nickel affinity chromatography.226site of Asp is located in the “NEEDH” that is a highly conserved negativecharge-intensive areas, which plays an important role in the catalytic reaction of thisregion. By enzyme activity tests showed that only mutant D226E has remains9.8%ofthe wild-type AK activity, and its K_m^ATPand K_m^Argare more increased than wild-typeAK, while the D226N and D226A mutant almost completely lost all activity. Byfluorescence spectroscopy and circular dichroism spectra the differences between wildtype and mutant AK were compared, the two conformational changes greatly. By theenzyme and substrate binding analysis shows that the mutant still has the function ofinduced conformational change binding with the corresponding substrate. Thereforespeculate that the Asp226plays an important role in supporting the stability of AKstructure and function.Tyr89and Arg330, respectively, which are highly conserved two sites in theN-terminal domain and the C-terminal domain of AK, they were linked togetherthrough hydrogen bonds. The differences between wild type and mutant AK has beencompared by determination of enzyme activity, fluorescence spectrometry andthermal stability analysis of the enzyme. The results are that the mutant Y89F andR330K only, respectively, remained the56.2%and23.4%activity of wild-type AK, the rest of the mutants almost have no activity, and found in the same purification andstorage conditions, the mutant R330K, R330L and the double mutant Y89F/R330L areeasily oxidized. The conformation of the mutant and wild type were also significantlydifferent and thermal stability of R330K were the strongest, Y89F were the weakest.89Tyr and330Arg to maintain the integrity of the AK structure and function onlythrough the interaction of hydrogen bonds were believed.