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J. Biol. Chem., Vol. 276, Issue 13, 10103-10109, March 30, 2001

This Article Full Text Full Text (PDF) All Versions of this Article: 276/13/10103    most recent M008828200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hitomi, K. Articles by Todo, T. Search for Related Content PubMed PubMed Citation Articles by Hitomi, K. Articles by Todo, T.

Role of Two Histidines in the (6-4) Photolyase Reaction^*

Kenichi Hitomi^§, Haruki Nakamura^¶, Sang-Tae Kim^§, Toshimi Mizukoshi, Tomoko Ishikawa^§, Shigenori Iwai, and Takeshi Todo^§

From the  Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, the ^§ Radiation Biology Center, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, and the ^¶ Laboratory of Protein Infomatics, Research Center for Structural Biology, Institute for Protein Research, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan

The reaction mechanism of Xenopus (6-4) photolyase was investigated using several mutant enzymes. In the active site, which is homologous between the cis,syn-cyclobutane pyrimidine dimer and (6-4) photolyases, four amino acid residues that are specific to (6-4) 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 (6-4) photoproduct. Both the H354A and H358A mutations resulted in an almost complete loss of the repair activity, although the Trp²⁹¹ and Trp³⁹⁸ mutants retained some activity. Taking the pH profile of the (6-4) photolyase reaction into consideration with this observation, we propose a mechanism in which these histidines catalyze the formation of 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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