Biochemical and Structural studies on the Muscle- Specific Creatine Kinase of the Common Carp (Cyprinus carpio) at Low Temperature
Date Issued
2011
Date
2011
Author(s)
Wu, Chih-Lu
Abstract
Extreme environmental change is an immediately challenge all over the world. The cold fronts sweeping in the winter, which causes millions of losses in aquaculture, is a severe challenge of Taiwan aquaculture industry. Marine biologists have developed some techniques to minimize the economic loss based on the studies of molecular mechanism of fish in stress. To overcome the change of temperature, physiologically, teleost has developed lots of mechanism to avoid harmful damage of ambient environment. The physiological effects of low temperature have mainly focused on following issues: metabolic compensation, homeoviscous adaptation of biological membranes, and thermal hysteresis.
The common carp could live from 35 to 5 °C. Its muscle-specific creatine kinase (M-CK) could maintain enzymatic activity at temperature around 15 °C. The present studies focus on the three common carp M-CK sub-isoforms (M1-, M2- and M3-CK) which are important in energy homeostasis. Specific activities of the common carp M1-CK were 3 to 8-folds higher than specific activities of M3- and rabbit M-CK at temperatures below 15 °C and pHs above 7.7. KmPCr and KmADP of M1-CK were relatively stable at pHs between 7.1 to 8.0, 25 to 5 °C. Its calculated activation energy of catalysis (Ea) at pH 8.0 was lower than at pH 7.1. Circular dichroism spectroscopy results showed that changes in secondary structures of M1-CK at the pHs and temperatures under studied were much less than in the cases of rabbit muscle-specific creatine kinase (RM-CK) and M3-CK.
When glycine 268 in RM-CK was substituted with asparagine 268 as found in carp M1-CK, the RM-CK G286N mutant specific activity at pH 8.0, 10 °C was more than 2-fold higher than the wild-type RM-CK at the same condition. Kinetic studies showed that Km values of the RM-CK G268N mutant were similar to those of the RM-CK, yet circular dichroism spectrum showed that the overall secondary structures of the RM-CK G268N, at pH 8.0, 5 °C, was almost identical to the carp M1-CK enzyme. The X-ray crystal structure of the RM-CK G268N revealed that amino acid residues involved in substrate binding were closer to one another than in the native RM-CK, and the side chain of cysteine 283 in active site of the RM-CK G268N pointed away from the ADP binding site. At pH 7.4-8.0, 35-10 °C, with a smaller substrate, dADP, specific activities of the mutant enzyme were consistently higher than the RM-CK and more similar to the carp M1-CK.
Then, to study the changes in physico-biochemical properties caused by residue 268 in RM-CK and M1-CK at low temperature, six more mutants, aspartic acid 268, lysine 268 or leucine 268 of RM-CK and M1-CK were generated. The peptide fragments near the active site were found to be phosphorylated. The specific activity results showed that, as in the case of asparagine 268, the aspartic acid 268 and lysine 268 mutants exhibited higher specific activities at low temperature and at higher pH, but not the leucine 268 mutant. The lower hydrophobicity side chain of residue 268 may help the stability of enzyme in glycerol containing buffer.
To sum up, we have found out that, the M1-CK enzyme seems to have evolved to adapt to the synchronized changes in body temperature and intracellular pH of the common carp. The smaller active site of the RM-CK G268N mutant might be one of the reasons for M-CK to improve activity at low temperature. The kinetic results and glycerol influence results indicated that charged side chain of residue 268 of M-CK might cause changes in protein conformation by interacting with water, and decreasing hydrophobicity of M-CK which in turn decreased its instability at low temperature.
Subjects
Cold tolerance
Creatine kinase
Enzyme activity
X-ray crystallography
Hydrophobicity
SDGs
Type
thesis
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