Activity and function of rabbit muscle-specific creatine kinase at low temperature by mutation at gly268 to asn268.
Carp muscle-specific creatine kinase M1 isoenzyme (M1-CK) seems to have evolved to adapt to synchronized changes in body temperature and intracellular pH. When gly(268) in rabbit muscle-specific creatine kinase was substituted with asn(268) as found in carp M1-CK, the rabbit muscle-specific CK G286N mutant specific activity at pH 8.0 and 10°C was more than 2-fold higher than that in the wild-type rabbit enzyme. Kinetic studies showed that K(m) values of the rabbit CK G268N mutant were similar to those of the wild-type rabbit enzyme, yet circular dichroism spectra showed that the overall secondary structures of the mutant enzyme, at pH 8.0 and 5°C, were almost identical to the carp M1-CK enzyme. The X-ray diffraction pattern of the mutant enzyme crystal revealed that amino acid residues involved in substrate binding are closer to one another than in the rabbit enzyme, and the cysteine283 active site of the mutant enzyme points away from the ADP binding site. At pH 7.4-8.0 and 35-10°C, with a smaller substrate, dADP, specific activities of the mutant enzyme were consistently higher than the wild-type rabbit enzyme and more similar to the carp M1-CK enzyme. Thus, the smaller active site of the RM-CK G268N mutant may be one of the reasons for its improved activity at low temperature.
Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, 115, Taiwan.
Chih-Lu
CL
Wu
Ying-Hao
YH
Li
Hsi-Chieh
HC
Lin
Yi-Hang
YH
Yeh
Hong-Young
HY
Yan
Chwan-Deng
CD
Hsiao
Cho-Fat
CF
Hui
Jen-Leih
JL
Wu
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine / Q000235:genetics; D002347:Carps; D003080:Cold Temperature; D052283:Creatine Kinase, MM Form / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D029941:Fish Proteins / Q000737:chemistry; D005998:Glycine / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D011817:Rabbits; D017386:Sequence Homology, Amino Acid
D029941:Fish Proteins; D001216:Asparagine; D052283:Creatine Kinase, MM Form; D005998:Glycine