Role of two histidines in the (6-4) photolyase reaction.
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, Gln(288), His(354), Leu(355), and His(358), and two conserved tryptophans, Trp(291) and Trp(398), 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 Trp(291) and Trp(398) 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.
Hitomi
K
K
Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.
Nakamura
H
H
Kim
S T
ST
Mizukoshi
T
T
Ishikawa
T
T
Iwai
S
S
Todo
T
T
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004260:DNA Repair; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017666:Deuterium Oxide / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011522:Protons; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014981:Xenopus
D011522:Protons; D005973:Glutamine; D006639:Histidine; D014364:Tryptophan; D004255:Deoxyribodipyrimidine Photo-Lyase; D007930:Leucine; D017666:Deuterium Oxide; D000409:Alanine