title: The activity of carp muscle-specific creatine kinase at low temperature is enhanced by decreased hydrophobicity of residue 268.
abstract: Abstract The muscle-specific forms of creatine kinase in rabbit (RM-CK) and carp (M1-CK) exhibit different temperature-dependent functional properties. Replacing the glycine at residue 268 of RM-CK with asparagine increases the enzyme's activity at 10°C. In this study, we investigated how hydrophobicity of residue 268 affects the biochemical properties of RM-CK and M1-CK at low temperature. We generated three mutants of both RM-CK and M1-CK: Asp268, Lys268, and Leu268. The secondary structures of these mutants were similar, as revealed by their circular dichroism spectra. Similar to the Asn268 mutants, the Asp268 and Lys268 mutants of RM-CK and M1-CK exhibited higher specific activities at 10°C and pH 8.0. However, no such effect was observed for the RM-CK and M1-CK Leu268 mutants. While in the presence of cryoprotectant (sucrose or trehalose), the activities of wild-type RM-CK and M1-CK mutant enzymes with a hydrophobic residue at 268 were higher, and the effect was more profound at pH 8.0. It may be inferred that water molecules affect protein conformation around residue 268, thereby influencing protein stability at low temperature.
authors.affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Chih-Lu
authors.initials: CL
authors.lastname: Wu
authors.forename: Bing-Yi
authors.initials: BY
authors.lastname: Li
authors.forename: Jen-Leih
authors.initials: JL
authors.lastname: Wu
authors.forename: Cho-Fat
authors.initials: CF
authors.lastname: Hui
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002347:Carps; D002942:Circular Dichroism; D003080:Cold Temperature; D052283:Creatine Kinase, MM Form / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029941:Fish Proteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D016297:Mutagenesis, Site-Directed; D011817:Rabbits
chemical_list: D029941:Fish Proteins; D052283:Creatine Kinase, MM Form