Creatine kinase: the reactive cysteine is required for synergism but is nonessential for catalysis.
Chemical modification of rabbit muscle creatine kinase (CK) with thiol-specific reagents led to partial or complete inactivation of the enzyme. Using site-directed mutagenesis, we have substituted the corresponding reactive Cys278 in the chicken cardiac mitochondrial creatine kinase (Mib-CK) with either glycine, serine, alanine, asparagine, or aspartate. The resulting mutant Mib-CK enzymes showed qualitatively similar changes in their enzymatic properties. In both directions of the CK reaction, a shift of the pH optimum to lower values was observed. Mutant Mib-CKs were severalfold more sensitive to inhibition by free ADP in the reverse reaction (ATP synthesis) and to free ATP in the forward reaction (phosphocreatine synthesis). With the exception of C278D, all mutant enzymes were specifically activated by chloride and bromide anions. C278D and wild-type Mib-CK were significantly inhibited under the same conditions. At low chloride concentrations, the Vmax of C278D was about 12-fold higher than that of C278N. Thus, Cys278 probably provides a negative charge which is directly or indirectly involved in maximizing CK activity. Under near-optimal conditions in the reverse reaction, mutants C278G and C278S showed about an 11-fold increase in Km(PCr), but only 1.7- and 2.8-fold reductions in Vmax, respectively, compared to wild-type Mib-CK. Thus, the reactive cysteine clearly is not essential for catalysis. For rabbit muscle CK, substrate binding had been shown to be synergistic (i.e., Kd > Km). We confirmed this finding for wild-type Mib-CK by determining the Kd and Km values for both substrates in the forward reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Furter
R
R
Institute for Cell Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich.
Furter-Graves
E M
EM
Wallimann
T
T
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D002384:Catalysis; D002645:Chickens; D003402:Creatine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D007527:Isoenzymes; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000494:pharmacology; D010725:Phosphocreatine / Q000096:biosynthesis; D013379:Substrate Specificity
D007527:Isoenzymes; D009711:Nucleotides; D010725:Phosphocreatine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D003545:Cysteine