Date Published:
1975 Nov 20Abstract:
A purification procedure for creatine kinase (EC 2.7.3.2) from muscle of the monke35--170 muequiv H+/mg protein per min at 30 degrees C and a yield of approx. 0.5 g/kg muscle. Assuming equilibrium kinetics, synergistic binding of substrates at one catalytic site is found for both the forward and back reactions. Kinetic constants for the binding of each substrate to the free enzyme and the enzyme-second substrate complex are determined and are compared with those for the enzyme from other species. Inhibition by small anions is determined in the presence of different combinations of substrates and products. SO4(2-) inhibits by simple competitive inhibition and probably binds at the site of the transferrable phosphoryl group. Inhibition by NO3-, NO2-, SCN- and Cl- is more complex and these ions are suggested to mimic the transferrable phosphoryl group in a planar transition-state complex. These anions stabilize the dead-end complex, enzyme-creatine-MgADP, which lacks the transferable phosphoryl group. The effects of these anions on the dissociation constants of the enzyme-substrate complexes is reported and is in accord with the above hypothesis. The dead-end complex in the absence of anion does not protect the essential thiol group against inhibition by iodoacetamide. Addition of NO3- or Cl- to the dead-end complex or a substrate equilibrium mixture without anion confers protection. The essential thiol group is inhibited by iodoacetamide at a rate which is essentially independent of pH over the normal stability range of the enzyme. Contrary to our previous report this pH independence is not altered by the presence of dead-end complex, creatine plus MgADP, in the presence or absence of anion or in the presence of a substrate equilibrium mixture. It is inferred that the 'essential' thiol group of the monkey enzyme has essentially the same properties as that of the rabbit enzyme. In consequence, the inferences made about the role of this group based on our previous work on the monkey enzyme are no longer valid. The present findings are compatible with the essential thiol group playing a conformational role in the catalytic process.
