Identification of the creatine binding domain of creatine kinase by photoaffinity labeling.
A new photoaffinity probe with a benzophenone group, N-dibenzylphospho-N'-(4-benzoyl)-benzylguanidine (BzPG), has been synthesized on the basis on our previously described creatine kinase bisubstrate analog. BzPG is also a bisubstrate type analog whose photoinsertion is inhibited by the natural substrates of creatine kinase. When rabbit CK-MM is irradiated in the presence of BzPG then cleaved by CNBr, one labeled peptide can be purified by reverse phase HPLC and sequenced. This sequence of 31 amino acids (Ala30-Val60) contains a region which could be responsible for isoenzyme selectivity and another one just preceding the 11 amino acid peptide (Asp61-Thr70) very recently described as a putative creatine binding site. This second peptide was deduced from the comparison of 18 amino acid sequence alignments. We proposed the creatine binding site to be essentially a peptide from Lys39 to Val71.
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Laboratoire de Biochimie C, Hôpital Edouard Herriot, 5 place d'Arsonval, F-69437 Lyon Cedex 03, France.
Steghens
J P
JP
Henry
R
R
Doutheau
A
A
Collombel
C
C
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D003402:Creatine Kinase / Q000737:chemistry*; D003488:Cyanogen Bromide / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008027:Light; D008958:Models, Molecular; D015394:Molecular Structure; D018482:Muscle, Skeletal / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry; D019920:Photoaffinity Labels / Q000737:chemistry; D011817:Rabbits; D017421:Sequence Analysis; D013053:Spectrophotometry
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D003402:Creatine Kinase; D003488:Cyanogen Bromide