Towards creatine kinase aggregation due to the cysteine modification at the flexible active site and refolding pathway.
The dimeric native state of creatine kinase (CK) was aggregated at conspicuous levels during cysteine modification at the active site with using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) under a high enzyme concentration. Measuring the ANS-binding fluorescence revealed that the hydrophobic surface of CK was increased by cysteine modification due to the flexible active site, and this resulted in insoluble aggregation, probably via non-specific hydrophobic interactions. To determine whether the aggregates can be refolded, 3M guanidine hydrochloride (GdnHCl) was used to dissolve the aggregates into the denatured form. Refolding of the solubilized enzyme sample was then conducted, accompanied by deprivation of DTNB from the CK in the presence of DTT. As a result, CK was reactivated by up to 40% with partial recovery of the tertiary (78%) and secondary structures (77%). To further elucidate its kinetic refolding pathway, both time interval measurements and a continuous substrate reaction were performed. The results showed that the refolding behavior was similar to the manner of normal CK folding with respect to the following two-phase kinetic courses. Additionally, the rate constants for the dimerization of the unfolded CK were dependent on the enzyme concentration and this was irrespective to the DTT concentrations, suggesting the rate-limiting steps of CK reassociation. The present study will expand our insight into the flexibility of the enzyme active site, which might act as a risk factor for inducing the unfavorable aggregation and partial refolding pathway of CK, as well as inducing an intermediate-like state recovery from aggregation.
Mu
Hang
H
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, PR China.
Zhou
Sheng-Mei
SM
Yang
Jun-Mo
JM
Meng
Fan-Guo
FG
Park
Yong-Doo
YD
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003402:Creatine Kinase / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry*; D019281:Dimerization; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D008954:Models, Biological; D009132:Muscles / Q000201:enzymology; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D004228:Dithionitrobenzoic Acid; D003402:Creatine Kinase; D019791:Guanidine; D003545:Cysteine; D004229:Dithiothreitol