Mutation of the conserved Asp122 in the linker impedes creatine kinase reactivation and refolding.
Creatine kinase (CK), a key enzyme in maintaining the intracellular energetic homeostasis, contains two domains connected by a long linker. In this research,we found that the mutations of the conserved Asp122 in the linker slightly affected CK activity, structure and stability. The hydrogen bonding and the ion pair contributed 2-5 kJ/mol to the conformational stability of CK. Interestingly, the ability of CK reactivation from the denatured state was completely removed by the mutations. These results suggested that the electrostatic interactions were crucial to the action of the linker in CK reactivation.
Liu
Yan-Ming
YM
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China.
Feng
Shan
S
Ding
Xiao-Lan
XL
Kang
Chi-Fei
CF
Yan
Yong-Bin
YB
D019943:Amino Acid Substitution / Q000187:drug effects; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics*; D017124:Conserved Sequence; D003402:Creatine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D019791:Guanidine / Q000494:pharmacology; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013329:Structure-Activity Relationship; D013696:Temperature
D050505:Mutant Proteins; D001224:Aspartic Acid; D003402:Creatine Kinase; D019791:Guanidine