Structural Changes Induced by Substrates and Anions at the Active Site of Creatine Kinase

作者：George H. Reed, Mildred Cohn · 发表于：Journal of Biological Chemistry · 年份：1972 · DOI：10.1016/s0021-9258(19)45214-9 · 被引用次数：133 · 研究领域：Advanced MRI Techniques and Applications、Electron Spin Resonance Studies、Lanthanide and Transition Metal Complexes

Abstract Structural changes at the active site of creatine kinase which occur upon binding of substrates and inhibitory anions have been examined by electron paramagnetic resonance (EPR) spectroscopy and proton relaxation rate (PRR) studies of manganous complexes with the enzyme. EPR spectra for solutions of ternary complexes involving enzyme and Mn(II)-nucleotides are quite similar to those for the binary Mn(II)-nucleotide complexes in solution and in the solid state. However, there are significant changes in the EPR spectra upon addition of creatine to solutions of E-MnADP or to E-MnATP. The changes show that the binding of creatine induces a rearrangement at the active site which results in a more asymmetrical electronic environment for the Mn(II). Substitutions of ligand groups from the protein into the Mn(II) coordination sphere are suggested as a possible source of the spectral changes. Anions such as nitrate and chloride produce further changes in the EPR spectrum of E-Mn-ADP-creatine and decrease the PRR of water for this complex. The anions apparently bind at the vacant phosphoryl site in the abortive quaternary complex, and their binding induces additional structural changes. Considerable specificity is observed in the effectiveness of various anions on the EPR spectrum or on the PRR of water, nitrate inducing the maximal effect. Experiments with modified substrates show that even minor differences e.g. dADP versus ADP in the molecular structure of the substrates bo...