The Role of the Lysyl Residue at the Active Site of Creatine Kinase: NUCLEAR OVERHAUSER EFFECT STUDIES

Abstract

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(II), and ADP. Omission of either of the substrates, creatine or ADP, or of the activator Mg(II) results in a large decrease in the observed effect. Such observations support the concept of a unique active site conformation for the transition state complex which requires the binding of all the components of the reaction. To assign the INDOR peak at 2.6 ppm, creatine kinase which had been selectively dansylated at the essential lysyl residues was investigated. It was established by water proton relaxation rate measurements of Mn(II) complexes that the dissociation constants for ADP and creatine binding to the enzyme are not significantly altered by dansylation, although there were changes in the enhancement factors of the Mn(II) complexes. Formate peak intensity measurements indicated little, if any, NOE for complexes with the dansylated enzyme. On the basis of the chemical shift of the formate INDOR peak, lack of an NOE for the quaternary complex of dansylated creatine kinase, and previous data showing loss of enzymatic activity upon dansylation of a single lysyl residue per active site, it is proposed that the e-CH2 protons of the lysyl residue at the active site are responsible for the formate NOE. Consequently, it is inferred that the role of the lysyl residue is to bind the transferable phosphoryl group. The interaction serves to provide the proper orientation for the transfer, and the electron-withdrawing properties of the lysyl e-NH3+ group facilitate nucleophilic attack at the phosphorus by the creatine guanidino group.