Structural And Functional Research On Phosphagen Kinases

A mutant of dimeric rabbit muscle creatine kinase, in which six residues (residues 2-7) at the N-terminal were removed by the PCR method, was studied to assess the role of these residues in dimer cohesion and to determine the structural stability of the protein. The specific activity of the mutant was 70.39% of that of the wild-type CK, and the affinity for Mg-ATP and creatine kinase substrates was slightly reduced compared with the wild-type protein. The structural stability of the mutant was investigated by a comparative equilibrium urea denaturation study and a thermal denaturation study. The activity of the mutant was more sensitive during urea titration compared to the wild-type. The data acquired by intrinsic fluorescence and far-UV circular dichroism during urea unfolding indicated that the secondary and tertiary structures of the mutant were more stable than those of wild-type CK. Furthermore, results of ANS fluorescence demonstrated that the hydrophobic surface of the mutant CKND6 was more stable during urea titration. Data from size exclusion chromatography experiments indicated that deletion of the six N-terminal residues resulted in a relatively loose molecular structure, but the dissociation of the mutant CKND6 occurred later during the unfolding process than for wild-type CK. Consistent with this result, the differential scanning calorimetry profiles demonstrated that the thermal stability of the enzyme was increased by removal of the six N-terminal residues. We conclude that a more stable quaternary structure was obtained by deletion of the six residues from the N-terminal of wild-type CK.The arginine kinase gene of sea cucumber Stichopus japonicus was cloned and inserted into the prokaryotic expression plasmid pET-21b. The protein was expressed in a soluble and functional form in Escherichia coli and purified by Blue Sepharose CL-6B, DEAE-32, and Sephadex G-100 chromotography with a final yield of 83 mg per liter of LB medium. The specific activity, electrophoretic mobility, and isoelectric focusing were all identical with that of arginine kinase purified from sea cucumber muscle. The fluorescence emission spectrum of arginine kinase had a maximum fluorescence at wavelength of 330 nm upon excitation at 295nm. These results are the first report of this purified protein.Urea titration was used to study the inactivation and unfolding equilibrium of arginine kinase (AK) from sea cucumber stichopus japonicus. Both fluorescence spectral and circular dichroism (CD) spectral data indicated that an unfolding intermediate of AK existed in the presence of 1.0 M to 2.0 M urea. This was further supported by the results of size exclusion chromatography (SEC). Spectral data suggested that this unfolding intermediate shared many structural characteristics with the native form of AK including its secondary structure, tertiary structure as well as its tetra-structure. Furthermore, according to the residual activity curve, this unfolding intermediate form still retained its catalytic function although its activity was lower than native AK. Taken together, the results of our study give direct evidence that an intermediate with partial activity exists in unfolding equilibrium states of AK during urea titration. 　　Crystals of sea cucumber AK was growed in this research. Single crystal suitable for X-ray diffraction was screened. The dimensions of the unit cell were determined. The calculated Matthew's coefficient indicated that there are three AK moleculars present in one asymmetric unit.