Tryptophan residues of creatine kinase: a fluorescence study

Abstract

Spectroscopic studies of rabbit skeletal muscle creatine kinase (CPK) and its complexes with adenosine phosphates have long suggested the occurrence of a tryptophan residue at or near the coenzyme binding sites [Kagi, J. H. R., Li, T.-K., and Vallee, B. L. (1971) Biochemistry 10, 1007-1015; Price, N. C. (1972) FEBS Lett. 24, 21-23]. This conjecture was further supported by nuclear Overhauser effect (NOE) 1H NMR studies indicating through-space interactions between protons of the adenine ring of bound ADP and one or more aromatic side chains of the proteins [Vasak, M., Nagayama, K., Wuthrich, K., Mertens, M. L., and Kagi, J. H. R. (1979) Biochemistry 18, 5050-5055). Further evidence for a tryptophan residue in the environment of the active site has now been obtained by fluorescence-quenching studies using iodide and acrylamide as external quenchers. Thus, while by the addition of iodide the tyrptophan fluorescence of unliganded CPK is reduced to about 75% of the unquenched control, no such effect is manifested upon addition of this quencher to the CPK .cntdot. ATP and CPK .cntdot. ADP complexes. Similarly, the relative effectiveness of quenching of the CPK-coenzyme complexes by acrylamide is only about 60% of that measured in the unliganded enzyme. Both these data and the spectral characteristics of the quenched fluorescence suggest that coenzyme binding perturbs a tryptophan residue that is close to the active site and that is partially exposed to the solent. The differential effectiveness of external quenchers on unliganded and liganded CPK allows the determination of the ligand binding equilibria by fluorescence-quenchability titration. The values obtained for complexes of CPK with ATP, ADP, and AMP are in good agreement with those obtained from other measurements.