Magnesium-adenosine diphosphate binding sites in wild-type creatine kinaseand in mutants: Role of aromatic residues probed by Raman and infrared spectroscopies

TWO distinct methods were used to investigate the role of Trp residues duri ng Mg-ADP binding to cytosolic creatine kinase (CK) from rabbit muscle: (1) Raman spectroscopy, which is very sensitive to the environment of aromatic side-chain residues, and (2) reaction-induced infrared difference spectros copy (RIDS) and photolabile substrate (ADP[Et(PhNO2)]), combined with site- directed mutagenesis on the four Trp residues of CK. Our Raman results indi cated that the environment of Trp and of Tyr were not affected during Mg-AD P binding to CK. Analysis of RIDS of wild-type CK, inactive W227Y, and acti ve W210,217,272Y mutants suggested that Trp227 was not involved in the stac king interactions. Results are consistent with Trp227 being essential to pr event water molecules from entering in the active site [as suggested by Gro ss, M., Furter-Graves, E. M., Wallimann, T., Eppenberger, Il. M., and Furte r, R. (1994) Protein Sci. 3, 1058-1068] and that another Trp could in addit ion help to steer the nucleotide in the binding site, although it is not es sential for the activity of CK. Raman and infrared spectra indicated that M g-ADP binding does not involve large secondary structure changes. Only 3-4 residues absorbing in the amide I region are directly implicated in the Mg- ADP binding (corresponding to secondary structure changes less than 1%), su ggesting that movement of protein domains due to Mg-nucleotide binding do n ot promote large secondary structure changes.