Activity of the common carp muscle form creatine kinase at low temperature depends on residue 268 with polar side chain
Date Issued
2011
Date
2011
Author(s)
Lin, Hsi-Chieh
Abstract
The common carp (Cyprinus carpio) required to maintain physiologic and metabolic capabilities when temperature of their habitat varies over a large temperature range 5 °C to 35 °C. At this broad range of temperature, ATP production in energy demanding cells is an important criterion to maintain the common carp. Creatine kinase is an important energy homeostasis enzyme. Muscle-specific creatine kinase (M-CK) catalyses the reversible reaction that converts phosphocreatine and ADP to creatine and ATP and thus provides ATP for muscle contraction. Previously, three common carp CK isoforms (M1-, M2- and M3-CK) were cloned. M1-CK has been suggested to have evolved to become the only CK that functions over the ranges of intracellular pH and body temperature variation that occurs in the common carp. The primary structures of RM-CK (M-CK of homeothermal rabbit) and carp M1-CK share 86% identity. By comparing the differences between RM-CK and M1-CK, and with RM-CK cDNA as template, RM-CK G268N show higher specific activity than wild type at pH 8.0 at 10 °C. This result suggests N268 residue plays an important role in conferring the appropriate 3D structure to M1-CK to function at low temperature. Therefore, we mutated 268 residues to aspartic acid, lysine or leucine and examined their properties. The mutants with polar side chain at residue 268 had higher activity and could maintain their function at low temperature. From the thermal stability, the polar mutants were more stable than non-polar mutants. Examining the structure surface of these mutants, when residue 268 was hydrophilic, it could connect nearby hydrophilic residues to form continuous hydrophilic region. This hydrophilic region could hydrogen bond with water, which then stabilizes the enzyme and results in maintaining activity at low temperature.
Subjects
common carp
low temperature
muscle-specific creatine kinase
Type
thesis
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