The Role of the Lysyl Residue at the Active Site of Creatine Kinase

作者：Thomas Leroy James, Mildred Cohn · 发表于：Journal of Biological Chemistry · 年份：1974 · DOI：10.1016/s0021-9258(19)42773-7 · 被引用次数：76 · 研究领域：Advanced MRI Techniques and Applications、Advanced NMR Techniques and Applications、NMR spectroscopy and applications

Considerable evidence from previous work supports the concept that formate is one of several planar anions which may occupy the binding site of the transferable phosphoryl group on creatine kinase, thus transforming the enzyme-Mg-(II)-ADP-creatine complex into an analog of the transition state of the active complex. Two NMR double resonance techniques, internuclear double resonance (INDOR) spectroscopy and intermolecular nuclear Overhauser effect (NOE) measurements of the formate proton, were employed to identify the binding site of formate on creatine kinase. The formate INDOR spectrum has a single peak at 2.6 ppm downfield from 2,2-dimethyl-2-silapentane-5-sulfonate due to dipole-dipole interaction of the formate proton with specific protons on the enzyme. The interacting protons are in close proximity to the formate proton and are characterized by a chemical shift of 2.6 ppm. Application of a strong radiofrequency field at 2.6 ppm results in a decrease in the intensity of the observed formate proton NMR peak, i.e. a negative NOE, for the transition state analog complex. The magnitude of the formate NOE depends on the formate to creatine kinase ratio, indicative of rapid exchange of formate between bound and free sites. Formate and nitrate compete for binding at the active site as shown by titration of the formate NOE with nitrate in the formate transition state analog complex. The large negative NOE for formate is obtained only in the case of the complex with creatine, Mg(...