NUCLEOTIDE-BINDING SITES IN WILD-TYPE CREATINE-KINASE AND IN W227Y MUTANT PROBED BY PHOTOCHEMICAL RELEASE OF NUCLEOTIDES AND INFRARED DIFFERENCE SPECTROSCOPY

Structural changes induced by nucleotide binding to the wild-type rabb it muscle creatine kinase (CK) and to its W227Y mutant were compared a nd probed by reaction-induced difference spectroscopy (RIDS). The reac tion was induced by the photorelease of nucleotide from the caged nucl eotides ADP[Et(PhNO2)] or ATP[Et(PhNO2)], producing the RIDS of CK. Th e concomitant addition of a saturated concentration of nucleotide and caged nucleotide modified the RIDS of CK, permitting structural change s caused by nucleotide binding in the wild-type creatine kinase to be identified. The W227Y mutant was inactive and its nucleotide binding s ite was partially impaired as shown by the disappearance or decrease o f several nucleotide-sensitive bands in the RIDS of W227Y mutant. The magnitude of the decrease was not the same for each band, suggesting t hat distinct groups of W227Y mutant were affected differently during n ucleotide binding. More precisely, the binding sites for gamma-phospha te and beta-phosphate of the nucleotide were not accessible in W227Y m utant as shown by the absence of the phosphate-sensitive 1666-1667-cm( -1) and 1625-cm(-1) bands in the RIDS of W227Y mutant. However the bin ding site of other parts of the nucleotide was partially accessible, s ince the 1638-1639-cm(-1) phosphate-insensitive band did not completel y vanish in the RIDS of W227Y mutant. The RIDS of W227Y mutant with AD P[Et(PhNO2)] and creatine lacked the 1613-cm(-1) and 1581-cm(-1) bands , associated with vibrational modes of creatine, suggesting that coupl ing between the binding sites of the nucleotide and of creatine was al tered in W227Y mutant. These results are in accordance with the earlie r suggestions that residue W227 in CK is essential for preventing wate r molecules from penetrating into the active site and for orienting nu cleotide in the binding site, by forming stacking interactions between its indole group and purine of the nucleotide and its indole group.