Characterization of the Active Site Structures of Arginine Kinase-Substrate Complexes: WATER PROTON MAGNETIC RELAXATION RATES AND ELECTRON PARAMAGNETIC RESONANCE SPECTRA OF MANGANOUS-ENZYME COMPLEXES WITH SUBSTRATES AND OF A TRANSITION STATE ANALOG

Abstract

Abstract The apparent number, n, of water ligands coordinated to bound Mn(II) in the ternary arginine kinase-MnADP or ATP complexes, in the abortive quaternary complex E-MnADP-arginine and in the transition state analog complex formed by addition of nitrate to the quaternary complex was estimated from the frequency dependence of the longitudinal proton relaxation rate of water (PRR) of lobster (Homarus americanus) muscle arginine kinase complexes. The apparent number of residual water ligands becomes progressively smaller as successive sites on the enzyme become occupied and is approximately 2 for the ternary nucleotide complexes, 1 for the abortive quaternary complex and considerably less than 1 for the transition state analog complex. The low values of n may be ascribed to the successive substitution of water ligands by protein ligands as each substrate and anion is successively bound to the enzyme or alternatively to an apparent rather than real disappearance of water ligands. The latter explanation implies that conformational changes induced by successive occupation of the binding sites on the enzyme are of such a nature that the rate of exchange of Mn(II)-bound water with solvent water becomes so slow for some Mn(II) water ligands that only a fraction of the total Mn(II)-bound water exchanges sufficiently rapidly to contribute to the PRR of bulk solvent. From the frequency dependence of the PRR of the ternary and quaternary complexes, it has been established that T1e, the electron spin relaxation time, is the dominant term in the correlation time which modulates the interaction between the Mn(II) electron spin and the water proton nuclear spin. Values of the electron spin relaxation times, T1e, estimated from PRR experiments are consistent with the lower limits of T1e obtained from line widths of the EPR spectra of the same complexes. No change is observed in the position of the peaks in the EPR spectra in proceeding from the binary MnADP complex to the ternary E-MnADP to the abortive quaternary E-MnADP-arginine or from MnATP to E-MnATP, and it may be inferred that there has been no gross modification in the symmetry of the electronic environment of Mn(II). Small changes in the line widths of the EPR spectra for these complexes do indicate that the accessibility of the Mn(II) liganded structure to collision with solvent molecules has been modified consistent with changes in the number of exchangeable water protons determined from PRR. Addition of nitrate to the abortive quaternary complex to form the transition state analog, as in the case of rabbit muscle creatine kinase, induces significant changes in the active site structure of arginine kinase as reflected in the altered EPR line shape. In this case altered line positions of the EPR spectrum also are observed. The latter indicates an asymmetry of the electronic environment of Mn(II) which may result from a substitution of ligands. The corresponding transition state analog for creatine kinase exhibits a much more anisotropic EPR spectrum. The EPR spectra are much more sensitive than PRR values to subtle changes in active site structure, e.g. spectral changes induced by d-arginine differ markedly from those produced by l-arginine in the presence of nitrate. Thiocyanate, nitrite, and formate at relatively high concentrations also modified the active site structure of arginine kinase, as evidenced by the alterations in the EPR spectrum of the quaternary complex upon addition of these anions. Modifications of the EPR spectra caused by these anions were qualitatively similar to those of nitrate but produced smaller changes in peak positions.