Abstract
Creatine kinase from rabbit muscle has been specifically labeled with a derivative of a nitroxide free radical on the two essential sulfhydryl groups, one per active site. The conformation in the environment of the enzyme-bound free radical and the change in conformation induced upon binding of the substrate, metal adenosine diphosphate, has been monitored by two parameters of the bound spin label, its electron paramagnetic resonance spectrum and its contribution to the proton relaxation rate of water. The specificity in activation of the enzymic reaction of the series of alkaline earth ions, Mg, Ca, Sr, Ba, was not reflected by differences in conformation of the ternary enzyme metal substrate complexes observable with the spin probe. However, with the inhibitory zinc ion, the conformation of ternary complex was distinctly different. The electron paramagnetic resonance spectra of complexes with two paramagnetic probes, namely, spin-labeled enzyme with paramagnetic metal adenosine diphosphate complexes (Mn(II), Ni(II), and Co(II)) revealed a spin-spin interaction between the protein-bound free radical and the paramagnetic metal ions. From the magnitude of the interaction, the distance between the manganous ion and the electron spin of the nitroxide moiety in the MnADP-spin-labeled creatine kinase was estimated to be in the range of 7-10 Å.
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