Nucleotide binding sites in wild-type creatine kinase and in W227Y mutant probed by photochemical release of nucleotides and infrared difference spectroscopy.

Abstract

Structural changes induced by nucleotide binding to the wild-type rabbit muscle creatine kinase (CK) and to its W227Y mutant were compared and probed by reaction-induced difference spectroscopy (RIDS). The reaction was induced by the photorelease of nucleotide from the caged nucleotides ADP[Et(PhNO2)] or ATP[Et(PhNO2)], producing the RIDS of CK. The concomitant addition of a saturated concentration of nucleotide and caged nucleotide modified the RIDS of CK, permitting structural changes caused by nucleotide binding in the wild-type creatine kinase to be identified. The W227Y mutant was inactive and its nucleotide binding site was partially impaired as shown by the disappearance or decrease of several nucleotide-sensitive bands in the RIDS of W227Y mutant. The magnitude of the decrease was not the same for each band, suggesting that distinct groups of W227Y mutant were affected differently during nucleotide binding. More precisely, the binding sites for gamma-phosphate and beta-phosphate of the nucleotide were not accessible in W227Y mutant 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 binding site of other parts of the nucleotide was partially accessible, since the 1638-1639-cm(-1) phosphate-insensitive band did not completely vanish in the RIDS of W227Y mutant. The RIDS of W227Y mutant with ADP[Et(PhNO2)] and creatine lacked the 1613-cm(-1) and 1581-cm(-1) bands, associated with vibrational modes of creatine, suggesting that coupling between the binding sites of the nucleotide and of creatine was altered in W227Y mutant. These results are in accordance with the earlier suggestions that residue W227 in CK is essential for preventing water molecules from penetrating into the active site and for orienting nucleotide in the binding site, by forming stacking interactions between its indole group and purine of the nucleotide and its indole group.