CREATINE-KINASE - NUCLEAR MAGNETIC-RESONANCE AND FLUORESCENCE EVIDENCE FOR INTERACTION OF ADENOSINE 5'-DIPHOSPHATE WITH AROMATIC RESIDUE(S)

The structural features of the purine binding site of creatine kinase (CPK) were explored by 1H NMR spectroscopy at 360 MHz, using the measurement of “truncated driven nuclear Overhauser effects” (TOE). Irradiation of the adenine C-2 and C-8 proton resonances in the CPK-ADP complex by this technique resulted in the negative enhancement of a number of resonances of the protein (intermolecular NOE's). Two of the affected resonances coincide with the irradiation frequencies shown previously to induce negative intermolecular NOE's in the adenine C-2 proton resonance of bound ADP [James, T. L. (1976) Biochemistry 15, 4724], The occurrence of several NOE's in the aromatic region between 6.5 and 8.0 ppm is compatible with the location of one or more aromatic side chains near the adenine ring in the CPK-ADP complex. Independent evidence for an interaction between the purine moiety of the coenzyme and aromatic amino acid chromophores comes also from quenching studies of protein fluorescence. Binding of nucleoside phosphates reduces tryptophan emission of CPK. The extent of quenching by ADP and GDP corresponds to the relative magnitudes of the Forster overlap integrals, thus suggesting a resonance transfer mechanism. Since the calculated critical Forster distance for resonance transfer between ADP and the affected tryptophanyl residues in CPK is not larger than 5 Å, at least one tryptophanyl residue must be located in the immediate vicinity of the purine binding site of CPK. The data are in accordance with our previous proposal that the coenzyme-induced Cotton effects at 260 nm arise from a dipole-dipole interaction of the adenine transition with a nearby aromatic oscillator. © 1979, American Chemical Society. All rights reserved.