Role of two histidines in the (6-4) photolyase reaction

The reaction mechanism of Xenopus (6-4) photolyase was investigated using s everal mutant enzymes. In the active site, which is homologous between the cis,syncyclobutane 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 t he substrate, which suggested a hydrophobic interaction with the (6-4) phot oproduct. Both the H354A and H358A mutations resulted in an almost complete loss of the repair activity, although the Trp291 and Trp398 mutants retain ed some activity. Taking the pH profile of the (6-4) photolyase reaction in to consideration with this observation, we propose a mechanism in which the se histidines catalyze the formation of the four-membered ring intermediate in the repair process of this enzyme. When deuterium oxide was used as a s olvent, the repair activity was decreased. The proton transfer shown by thi s isotope effect supports the proposed mechanism. The substrate binding and the reaction mechanism are discussed in detail using a molecular model.