Studies On The Refolding And Part Structure Of Arginine Kinase

Arginine kinase (ATP: L-arginine phosphotransferase EC 2.7.3.3), plays an importantrole in cellular energy metabolism in invertebrates. Arginine kinase from ShrimpFeneropenaeus Chinensis (40 kDa) was taken a model protein to study the refoldingprocess and its partial structure. In dilute denaturant, two equilibrium refolding intermediates (I and N′) werediscovered, and a refolding scheme of urea-denatured AK was proposed. The first foldingintermediate (I) was inactive one in urea solution ranging between 2.4 and 3.0 M. Thesecond (N′) existed between 0.4 and 0.8 M urea solution, with slightly increased activity.The refolding kinetics showed that the urea-denatured AK was in two-phase refolding.Proline isomerization occurred in the unfolding course of AK, which blocked the slow phaseof refolding. The activity recovery of GduCl-denatured AK is very low and dependent on theprotein concentration in the process of refolding. The nonionic detergent Triton X-100 andTween 20 (≤100mM) not only effectively blocked the aggregation but also enabled thedenatured AK to recover most of its native activity,and the aggregation could be partlydissolved in Tween 20 and Triton X-10, which could refold into a native arginine kinasestate and lead to the enhancement of activity recovery. The apparent activity recovery had acooperative relationship with detergents in the process of refolding, which suggested theexistence of some interaction between the detergent and the refolding intermediate. On thebasis of the study results, a scheme of refolding was proposed. The reduction of 5, 5'-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase bydithiothreitol has been studied using the kinetic theory of the substrate reaction duringmodification of enzyme activity. The results show that the reactive cysteine of arginine kinasemay play an important role not in the binding to the transition-state analogue but in theconformational changes caused by the transition-state analogue. The excess molecular oxidized dithiothreitol could result in a loss of activity and - II -Abstractconformational change of arginine kinase. The results of size exclusion chromatography andsodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that no disulfide bondwas formed among the protein molecules in the oxidized-DTT solution.