ATP binding site of mitochondrial creatine kinase. Affinity labelling of Asp-335 with C1RATP.
The ATP binding site of mitochondrial creatine kinase from chicken heart has been studied by modifying the purified enzyme with a 14C-labelled ATP analogue, C1RATP, in which the reactive label was covalently bound to the gamma-phosphate group of ATP. The modified enzyme was digested by pepsin, and a single radioactive nonapeptide was isolated by HPLC. Amino acid analysis and direct sequence determination revealed that the isolated peptide corresponds to amino acids 335-343 within the C-terminal region of Mi-CK, this peptide being highly preserved throughout evolution. Asp-335 is very likely the site of modification by C1RATP. The specificity of the ATP analogue for the active site of creatine kinase was demonstrated by the inhibition of the enzymatic activity of Mi-CK by C1RATP and by the prevention of this inhibition bij ADP.
James
P
P
Laboratory of Biochemistry, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zürich.
Wyss
M
M
Lutsenko
S
S
Wallimann
T
T
Carafoli
E
E
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D002645:Chickens; D003402:Creatine Kinase / Q000378:metabolism*; D007527:Isoenzymes; D008929:Mitochondria, Heart / Q000378:metabolism*; D008969:Molecular Sequence Data; D010434:Pepsin A; D010446:Peptide Fragments / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid
D000345:Affinity Labels; D007527:Isoenzymes; D010446:Peptide Fragments; D001224:Aspartic Acid; C025165:ATP-gamma-4-(N-2-chloroethyl-N-methylamino)benzylamidate; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D010434:Pepsin A