Mutation of conserved active-site threonine residues in creatine kinase affects autophosphorylation and enzyme kinetics.
Muscle-type creatine kinase (MM-CK) is a member of an isoenzyme family with key functions in cellular energetics. It has become a matter of debate whether the enzyme is autophosphorylated, as reported earlier [Hemmer, Furter-Graves, Frank, Wallimann and Furter (1995) Biochim. Biophys. Acta 1251, 81-90], or exclusively nucleotidylated. In the present paper, we demonstrate unambiguously that CK is indeed autophosphorylated. However, this autophosphorylation is not solely responsible for the observed microheterogeneity of MM-CK on two-dimensional isoelectric focusing gels. Using phosphoamino-acid analysis of (32)P-labelled CK isoforms, phosphothreonine (P-Thr) residues were identified as the only product of autophosphorylation for all CK isoenzymes. The phosphorylated residues in chicken MM-CK were allocated to a region in the vicinity of the active site, where five putative phosphorylation sites were identified. Site-directed threonine-valine-replacement mutants reveal that autophosphorylation is not specific for one particular residue but occurs at all examined threonine residues. The enzyme kinetic parameters indicate that the autophosphorylation of CK exerts a modulatory effect on substrate binding and the equilibrium constant, rather than on the catalytic mechanism itself.
Stolz
Martin
M
Blood Transfusion Service SRC Bern Ltd., PCR diagnostics, Murtenstrasse 133, 3008 Bern, Switzerland.
Hornemann
Thorsten
T
Schlattner
Uwe
U
Wallimann
Theo
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002855:Chromatography, Thin Layer; D003402:Creatine Kinase / Q000235:genetics* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011817:Rabbits; D013329:Structure-Activity Relationship; D013912:Threonine / Q000235:genetics* / Q000378:metabolism
D007527:Isoenzymes; D013912:Threonine; D003402:Creatine Kinase