Creatine kinase. Modification of the working enzyme

Abstract

Protection against inhibition of creatine kinase [EC 2.7.3.2] by iodocetamide is measured by the decrease in the rate constant for the inhibition reaction. A mixture of purified substrates at equilibrium protects strongly when all the components of the mixture are nearly saturating. The protection by substrate working in the forward direction only (from creatine and MgATP) was measured by performing the experiment rapidly at low enzyme concentrations. Varying the concentration of substrate showed that the amount of protection, when the substrates of the forward reaction are saturating is about 80% (100% protection would imply a value of zero for the inhibition reaction). The effects of Ca2+ and Mg2+ are compared. The complex creatine-NO3-MgADP, which is considered to be either a transition-state analog or an analog of an intermediate in the reaction pathway, protects fully against iodoacetamide. Creatine and MgADP alone, or together without NO3-, do not protect. The degree of protection by the working enzyme may represent the proportion of enzyme molecules having a conformation complementary to a creatine-PO3-MgADP intermediate.