Initial Study On The Role Of Reactive Cysteine In Arginine Kinase From Sea Cucumber Stichopus Japonicus

The studies focused on the role of cysteine residues in dimeric argininekinase (AK, EC2.7.3.3) from sea cucumber Stichopus japonicus with chemicalmodification and reactivation kinetic.The mutation gene with His-Tag in C terminal,AK-C274AH,wasobtained in this paper. The protein was expressed in a soluble and functionalfrom Escherichia coli and purified by Ni-NTA with the final yield of 150 mgper liter of LB medium, and the purity was 99%. The difficulty of purificationwas solved.There are ten cysteine residues in dimeric enzyme, but all of them werein the form of –SH from NR/R SDS-PAGE, unlike the dimeric CK. Thisindicated that the distances of thiol groups were too far or their orientationswere not proper to form disulfide bonds.The modification courses of native AK and mutation AK-C274AH werecompared with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The course ofnative AK is biphasic, and that of the mutation is monophasic. Theinactivation of native enzyme with DTNB is monophasic. The DTNBmolecules are accessible to about six cysteine residues for native AK (four formutation), among them two for quick phase (one for per subunit) and the otherfour for slow phase. The results explicitly suggest that Cys-274 is exposed toDTNB and reacts with DTNB faster than other accessible cysteine residueswith the loss of enzyme activity. The reactivation courses of DTNB-modified AK by dithiothreitolwere investigated on the basis of the kinetic theory of the substrate reactionduring the modification of enzyme activity. The modified AK can bereactivated by an excess concentration of dithiothreitol in a monophasickinetic course. The results show that the reactive Cys-274 is near the ATPbinding site. The presence of ATP or the transition-state analog markedlyslows the apparent reactivation rate constant. The analog components,arginine-ADP-Mg2+ can induce conformational changes of the modifiedenzyme and mutation enzyme, but adding NO3-can not induce their furtherchanges that occur on the native enzyme. The location and the role of reactiveCys-274 in the catalysis of AK are discussed. The results suggest that reactiveCys-274 may be located in the hinge area of the two domains of AK, and itmay play an important role not in the binding of the transition-state analog butin the conformational changes caused by the transition-state analog.