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.

The apparent number, n, of water ligands coordinated to bound Mn(I1) in the ternary arginine kinase-MnADP or ATP complexes, in the abortive quatemary complex E-MnADParginine and in the transition state analog complex formed by addition of nitrate to the quatemary 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 quatemary complex and considerably less than 1 for the transition state analog complex. The low values of R 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(I1) 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 quatemary complexes, it has been established that T1,, the electron spin relaxation time, is the dominant term in the correlation time which modulates the interaction between the Mn(I1) electron spin and the water proton nuclear spin. Values of the electron spin relaxation times, Tie, estimated from PRR experiments are consistent with the lower limits of TI, obtained from line widths of the EPR spectra of the same complexes.