Electron Paramagnetic Resonance and Proton Relaxation Rate Studies of Spin-labeled Creatine Kinase and Its Complexes

Abstract

Abstract A spin-labeled derivative of rabbit muscle creatine kinase has been prepared by reaction of the essential sulfhydryl group at the active sites with a stoichiometric amount of the nitroxide radical N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) iodoacetamide. The spin-labeled enzyme retains the ability to bind nucleotide substrates although it lacks the phosphotransferase and ATPase activities of the native enzyme. Two magnetic resonance properties of the specifically spin-labeled enzyme and its substrate complexes were used to probe the local environment of the covalently bound radical: (a) the electron paramagnetic resonance (EPR) spectrum and (b) the enhancement of the proton relaxation rate of water protons. The protein-bound radical shows a broad, asymmetric EPR spectrum resembling a solid state powder spectrum; the outer peaks are separated by 62 gauss compared with 31 gauss for the free radical and the amplitude of the central peak is 14 times greater in the unbound than the bound spin label. In addition, the spin-labeled protein enhances the water proton relaxation rate by approximately 1 order of magnitude. Both phenomena indicate a high degree of immobilization of the bound spin label. Metal nucleotide binding increases immobilization of the spin label as evidenced by the EPR spectrum. The spectral changes represent the first direct evidence for a proposed conformational change in the region of the active site on addition of divalent ions to the enzyme-nucleotide binary complex. The proton relaxation rate of water is even more sensitive to local structural changes and reveals differences between the enzyme and its binary nucleotide complexes as well as between binary and ternary enzyme-metal nucleotide complexes. Creatine alone or in combination with metal nucleotides fails to affect either the EPR spectrum or the water proton relaxation rate of the spin-labeled enzyme. The nucleotide complexes of the alkali earth cations, Mg(II), Ca(II), Sr(II), and Ba(II), produce approximately the same changes in the EPR of the spin-labeled enzyme, amounting to a diminution of 20% in central peak amplitude upon addition of metal-ADP and a somewhat smaller effect with the ATP complexes. Quantitative differences in the properties of the different metal complexes are most easily observed by measurement of the proton relaxation rate of water under optimal temperature and frequency conditions. The ternary complexes of the irreversibly inhibitory Zn(II) nucleotides with spin-labeled enzyme have qualitatively different EPR spectra than do the corresponding complexes of alkali earth ions. Apparent dissociation constants have been calculated from both EPR and proton relaxation rate titrations for the dissociation of the alkali earth nucleotides from the ternary complexes. All are of the same order of magnitude as for the native enzyme, about 0.1 mm for ADP ternary complexes.