Photoaffinity Labelling of Arginine Kinase and Creatine Kinase with a gamma-P-Substituted Arylazido Analogue of ATP

Abstract

An ATP analog with a photoactivated azide group attached to the .gamma.-phosphate via an amide bond, ATP .gamma.-p-azidoanilide, appeared to have potential use as a photoaffinity label for the nucleotide-binding regions of ATP:guanidine phosphotransferases. Upon photolysis in the presence of lobster muscle arginine kinase and rabbit muscle creatine kinase, the analog is converted to a potent inhibitor of these 2 kinases. This photo-dependent inhibition is specific as it cannot be induced by azidoaniline, a mixture of azidoaniline and ATP or by ATP .gamma.-p-aminoanilide. Pre-irradiated under suitable conditions, the photoanalog still shows a transitory inhibitory effect which slowly vanishes with time (to.5 = 3 h). The photoinhibition is significantly decreased by the presence of ATP or ADP but is completely prevented by the addition of a mixture of nucleotide and guanidine substrates. Differential spectroscopy and affinity chromatography on Sepharose-ATP demonstrated the inability of photoinactivated arginine kinase and creatine kinase to recognize their nucleotide substrates. Experiments with [14C]ATP .gamma.-p-azidoanilide indicated that photolysis is associated with an irreversible and stoichiometric binding of the ATP analog to the enzymes. Autoradiographs made with the peptide maps corresponding to the tryptic digests of each 14C-labeled photomodified enzyme showed an unexpected highly specific labeling of the proteins. Thiol titrations of the kinases which were subjected to various photolysis conditions led to the conclusion that arylnitrene moiety of the photoanalog is covalently attached to the single reactive cysteinyl side chain present in the active-site region of the 2 homologous kinases. This amino acid residue is apparently located near the phosphate chain binding subsite occupied by the ATP analog and probably also by the natural nucleotide substrates.