Non-identical behaviour of the subunits of rabbit muscule creatine kinase.
1. The dimeric enzyme creatine kinase (ATP: creatine N-phospotransferase, EC 2.7.3.2) from rabbit muscle was reacted with three separate reagents, each of which specifically modifies one thiol group per subunit. 2. The reactions of the enzyme with these reagents (4-chloro-7-nitrobenzofurazan, 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetate) all behave as normal second-order processes. This indicates that the thiol groups on the two subunits of the enzyme react at the same rate as each other in all three cases. 3. The effects of various ligands (Mg2+, ADP and creatine, and combinations of these) on the kinetics of the reactions were studied. In all cases the reactions behave as normal second-order processes. 4. In the presence of the ligand combination Mg2+ plus ADP plus creatine plus nitrate, which has been postulated to form a "transition state analogue" complex with the enzyme, the reactions of the thiol group show considerable deviation from second-order kinetics. This indicates that the thiol groups on the two subunits react at different rates from each other. A similar effect is also noted in the presence of the combination ADP plus creatine plus nitrate. 5. The binding of ADP to the enzyme (studied by equilibrium dialysis) is hyperbolic in the absence of other ligands or in the presence of Mg2+ or Mg2+ plus creatine. The dissociation constant is similar in all three cases. 6. In the presence of creatine plus nitrate (with or without Mg2+) the bindings of ADP to the enzyme is tightened considerably and the binding plots indicate the presence of either negative interactions between the subunits or two distinct types of binding sites. 7. Possible causes for the observed non-identical behaviour of the two subunits of the enzyme are discussed.
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D003402:Creatine Kinase / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D009132:Muscles / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis
D007461:Iodoacetates; D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D003402:Creatine Kinase; D009327:4-Chloro-7-nitrobenzofurazan