Studies On The Refolding And Structure Of Arginine Kinase From Sea Cucumber Stichopus Japonicus

Arginine kinase (AK) from sea cucumber was taken as a model protein to studythe enzyme catalysis mechanism and unfolding/refolding process of the oligomericprotein.Methods of chemical modification and site-directed mutagenesis were used inthis paper to investigate the active essential animo acids. The modification of AK byOPTA indicated an active essential pair of Cys-Lys near or at the active sites of AK.The active essential Cys was located in a highly hydrophobic environment,presumably near ATP and ADP binding region. Sequence comparison in thephosphagen kinase family suggested that this essential Cys maybe the conservativeCys-274. Roles of Trp-208 and Trp-218 of AK were individually investigated bysite-directed mutagenesis. Both residues were fully conserved in the phosphogenkinase family. Our studies revealed that Trp-218 was located at the active site of AKand it also took part in stabilizing the conformational structure of AK. Althoughanother tryptophane, Trp-208 was not located at the active sites, it may take part inmaintaining the correct dimer conformation for catalysis. In addition, Trp-208 alsoseemed to play an important role in correct protein folding.Dimeric AK was used as a model of oligomeric protein to investigate itsunfolding/refolding pathway in thermal or denaturant denaturation, with theaim to trap the possible equilibrium or kinetic intermediates. The thermaldenaturation was an irreversible process. After the dimeric AK dissociated tomonomer, the monomeric intermediate was so unstable that it rapidly formedthe soluble aggregation, and then the precipitation. In addition, the free Mg2+could accelerate this process. The unfolding of AK induced by GdnHCl wasdescribed as follows: N2?I1→I2?2U, involving at least two dimericintermediates. The refolding processes initiated by rapid dilution inrenaturation buffers indicate that there seems to be an energy barrier betweenthe two intermediates, which makes it difficult for AK denatured at highGdnHCl concentrationsto reconstitute and regain its catalytic activitycompletely. Osmolytes could help denaturated AK overcome this energybarrier and thus refold to its active conformation. Since there was an energybarrier in the refolding process of GdnHCl-denatured AK, which prevented usto gain more comprehensive knowledge about its refolding pathway, wechange the denaturant from GdnHCl to urea, investigating both of itsequilibrium and kinetic refolding process. Equilibrium research revealed thatthe refolding of urea-denatured AK involves at least one globular-like (I2) andone native-like (I1) dimer intermediate. Kinetics research suggested theexistence of a burst phase intermediate, which possessed the similarcharacteristics as I1 in equilibrium refolding.