The reaction of rabbit muscle creatine kinase with some derivatives of iodoacetamide.
The dimeric enzyme creatine kinase from rabbit muscle was treated with three derivatives of iodoacetamide that are capable of introducing fluorescent groups into the enzyme. All the three reagents (4-iodoacetamidosalicylate (IAS), 5-[N-(iodoacetamidoethyl)amino]-naphthalene-1-sulphonate (IAEDANS) and 6-(4-iodoacetamidophenyl)aminonaphthalene-2-sulphonate (IAANS)) were shown to react at the same single thiol group on each enzyme subunit, leading to complete inactivation of the enzyme. The reaction with IAS was extremely rapid by comparison with the reaction with iodoacetamide or iodoacetate, but various lines of evidence suggest that IAS is not a true affinity label. However, kinetic and binding studies indicate that salicylate itself probably binds at the nucleotide-binding site on the enzyme. As the size of the modifying reagent increased, the first thiol group reacted more rapidly than the second; this trend was more pronounced at 0 degree C than at 25 degree C. With the largest modifying reagent used (IAANS), the pronounced biphasic nature of the modification reaction permitted the preparation of a hybrid enzyme in which only one subunit was modified, but a study of the thiol-group reactivity showed that this hybrid enzyme preparation underwent subunit rearrangement.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003402:Creatine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007460:Iodoacetamide / Q000031:analogs & derivatives / Q000494:pharmacology*; D007461:Iodoacetates / Q000494:pharmacology*; D007700:Kinetics; D008024:Ligands; D009132:Muscles / Q000201:enzymology*; D011817:Rabbits; D012459:Salicylates / Q000494:pharmacology
D007461:Iodoacetates; D008024:Ligands; D012459:Salicylates; D000244:Adenosine Diphosphate; D003402:Creatine Kinase; D007460:Iodoacetamide