ADP-BINDING AND ATP-BINDING SITES IN NATIVE AND PROTEINASE-K-DIGESTEDCREATIVE KINASE, PROBED BY REACTION-INDUCED DIFFERENCE INFRARED-SPECTROSCOPY

Conformational changes induced by nucleotide binding to native creatin e kinase (CK) from rabbit muscle and to proteinase-K-digested (nicked) CK, were investigated by infrared spectroscopy. Photochemical release of ATP from ATP[Et(PhNO2)] in the presence of creatine and native CK produced reaction-induced difference infrared spectra (RIDS) of CK rel ated to structural changes of the enzyme that paralleled the reversibl e phosphoryl transfer from ATP to creatine. Similarly the photochemica l release of ADP from ADP[Et(PhNO2)] in the presence of phosphocreatin e and native CK allowed us to follow the backward reaction and its cor responding RIDS. Infrared spectra of native CK indicated that carboxyl ate groups of Asp or Glu, and some carbonyl groups of the peptide back bone are involved in the enzymatic reaction. Native and proteinase nic ked CK have similar Stokes' radii, tryptophan fluorescence, fluorescen ce fraction accessible to iodide, and far-ultraviolet CD spectra, indi cating that native and modified enzymes have the same quaternary struc tures. However, infrared data showed that the binding site of the gamm a-phosphate group of the nucleotide was affected in nicked CK compared with that of the native CK. Furthermore, the infrared absorptions ass ociated with ionized carboxylate groups of Asp or Glu amino acid resid ues were different in nicked CK and in native CK.