[Creatine kinase from rabbit skeletal muscles: formation of O-acyltyrosine as a result of the activation of the carboxylic group of the enzyme active site by affinity reagents, nucleotide imidazolides].
Creatine kinase from skeletal muscle (EC 2.7.3.2) was inactivated by means of imidazolides of AMP, ADP, ATP. Rates of the inactivation of the enzyme's M- and M'-subunits differ 50-100 fold and decrease in the presence of ADP and ATP. Differential spectrum of the native and modified enzymes corresponds to the spectrum of N,O-diacetyltyrosine. Kinetic curves of hydroxylamine-dependent destruction of N,O-diacetyltyrosine and of alteration of differential spectrum of the modified and native enzymes essentially coincide. The enzyme's inactivation appears to be caused mainly by the formation of a bond between nucleotide imidazolides activated carboxyl group of the active centre and OH-group of Tyr residue arranged in the close proximity. The stoichiometry of acyltyrosine formation is evaluated as 2.1 +/- 0.2 mole per mole of the functional dimer. Along with formation of ester bond between amino acid residues, a covalent attachment of 0.03-0.06 mole of [14C]nucleotides per mole of enzyme is observed. As the data of acid hydrolysis show, Im-ATP and Im-AMP block epsilon-amino group of Lys and guanidine group of Arg, respectively. Reasons of the multiple modification of creatine kinase by affinity reagents are discussed. The results obtained and literature data are summarised in the hypothetical scheme of disposition of various amino acid residues in the active centre of creatine kinase.
D000227:Adenine Nucleotides / Q000494:pharmacology*; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002264:Carboxylic Acids; D003402:Creatine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D007093:Imidazoles / Q000494:pharmacology*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D011817:Rabbits; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000227:Adenine Nucleotides; D000345:Affinity Labels; D002264:Carboxylic Acids; D007093:Imidazoles; C053151:N,O-diacetyltyrosine; D014443:Tyrosine; D003402:Creatine Kinase
Kreatinkinaza iz skeletnykh myshts krolika: obrazovanie O-atsiltirozina v rezul'tate aktivatsii karboksil'noĭ gruppy aktivnogo tsentra fermenta pod deĭstviem affinnykh reagentov-- imidazolidov nukleotidov.