Properties and reaction with iodoacetamide of adenosine 5'-triphosphate-creatine phosphotransferase from human skeletal muscle. Further evidence about the role of the essential thiol group in relation to the mechanism of action.
1. The purification of creatine kinase from human and monkey skeletal muscle by horizontal electrophoresis on Sephadex blocks is described. 2. The purified enzymes are shown to have similar chemical and kinetic properties to the rabbit muscle enzyme and a common mechanism is inferred. 3. Iodoacetamide has a similar apparent second-order inhibition constant with the human and rabbit enzymes, but the inhibition does not go to completion with the former. This is even more marked with the monkey enzyme, which has more reactive thiol groups, but inhibition is only about 50%. 4. Single substrates have little effect on the inhibition by iodoacetamide, but with the primate enzymes, in contrast with the rabbit enzyme, high concentrations of ADP-Mg(2+) plus creatine convert the essential thiol group from being pH-independent into one with a normal ionization. Low concentrations of ADP-Mg(2+) plus creatine first enhance the rate of inactivation, but cause protection as the reaction proceeds. These results are interpreted to indicate an activation of the thiol group on the subunit to which the substrates bind and a co-operatively induced decrease in the activity of the thiol group on the other subunit which lacks substrates. 5. The effects of a substrate equilibrium mixture on the rate of inhibition are essentially those of ADP-Mg(2+) plus creatine. 6. Since no substrate combination affords significant protection to the thiol group associated with the catalytic site to which the substrates are bound, it is concluded that any mechanism involving the thiol group in a direct participation in the transition-state complex of the catalytic reaction must be abandoned unless the transition state is only a small part of the time taken for one catalytic cycle.
D000255:Adenosine Triphosphate; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003402:Creatine Kinase / Q000032:analysis* / Q000037:antagonists & inhibitors; D004586:Electrophoresis; D000882:Haplorhini; D006801:Humans; D007461:Iodoacetates / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D009584:Nitrogen / Q000032:analysis; D011817:Rabbits; D014364:Tryptophan / Q000032:analysis; D014443:Tyrosine / Q000032:analysis
D000596:Amino Acids; D007461:Iodoacetates; D014443:Tyrosine; D014364:Tryptophan; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D008274:Magnesium; D009584:Nitrogen