Properties and mechanism of action of creatine kinase from ox smooth muscle. Anion effects compared with pyruvate kinase

1. An improved purification procedure for the brain-type creatine kinase from ox smooth muscle is described. 2. Michaelis constants show the characteristic dependence on the concentration of the second substrate: the derived constants are compared with those for the enzyme from ox brain. 3. Inhibition by iodoacetamide gives a biphasic curve and the total extent of the reaction depends on the enzyme concentration. The rate of inhibition at pH8.6 is not affected by creatine plus MgADP or by a range of simple anions. Addition of creatine plus MgADP plus either NO3- or Cl- ions affords 71.5 and 44% protection respectively. ADP could be replaced by 2-deoxy-ADP but not by αβ-methylene ADP, XDP, IDP, GDP or CDP. Nucleotides that did not protect would not act as substrates. 4. Difference-spectra measurements support the interpretation that addition of NO3- ions to the enzyme–creatine–MgADP complex causes further conformational changes in the enzyme accompanying the formation of a stable quaternary enzyme–creatine–NO3-–MgADP complex that simulates an intermediate stage in the transphosphorylation reaction. However, the enzyme structure is partially destabilized by quaternary-complex formation. IDP apparently fails to act as a substrate because it cannot induce the necessary conformational change. This behaviour is compared with that of rabbit skeletal muscle creatine kinase. 5. With pyruvate kinase from rabbit muscle, anions activate in the absence of an activating cation and either inhibit or have no effect in its presence. 6. Both activation and inhibition were competitive with respect to the substrate, phosphoenolpyruvate, and curved double-reciprocal plots were obtained. The results may be interpreted in terms of co-operatively induced conformational changes, and this is supported by difference-spectra measurements. However, the Hill coefficient of 1 was not significantly altered. 7. Inhibition by lactate plus pyruvate is less than additive, indicating that both bind to the same site on the enzyme, whereas that by lactate plus NO3- is additive, indicating binding at separate sites. It is inferred that a quaternary enzyme–pyruvate–NO3-–MgADP complex could form, but no evidence was obtained to suggest that it possessed special properties comparable with those found with creatine kinase. The implications of these findings for the unidirectional nature of the mechanism of pyruvate kinase is discussed. 8. Lactate or α-hydroxybutyrate could not act instead of pyruvate to form a stable quaternary complex, although both activate the K+-free enzyme. Only the former inhibits the K+-activated enzyme. The activating cation both lowers the Michaelis constant for phosphoenolpyruvate and tightens up the specificity of its binding site.