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Papers In Press, published online ahead of print December 21, 2000
J. Biol. Chem, 10.1074/jbc.M008828200
Submitted on September 27, 2000
Revised on November 30, 2000
Accepted on December 20, 2000
Radiation Biology Center, Kyoto University, Kyoto 606-8501
Corresponding Author: todo{at}house.rbc.kyoto-u.ac.jp
The reaction mechanism of Xenopus (6Ð4) photolyase was investigated using several mutant enzymes. In the active site, which is homologous between the CPD and (6Ð4) photolyases, four amino acid residues that are specific to (6Ð4) photolyase, Gln288, His354, Leu355, and His358, and two conserved tryptophans, Trp291 and Trp398, were substituted with alanine. Only the L355A mutant had a lower affinity for the substrate, which suggested a hydrophobic interaction with the (6Ð4) photoproduct. Both the H354A and H358A mutations resulted in an almost complete loss of the repair activity, although the Trp291 and Trp398 mutants retained a reduced activity. Taking the pH profile of the (6Ð4) photolyase reaction into consideration with this observation, we propose a mechanism in which these histidines act as an acid and a base to form the four-membered ring intermediate in the repair process of this enzyme. When deuterium oxide was used as a solvent, the repair activity was decreased. The proton transfer shown by this isotope effect supports the proposed mechanism. The substrate binding and the reaction mechanism are discussed in detail using a molecular model.
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