Document Detail


Role of two histidines in the (6-4) photolyase reaction.
MedLine Citation:
PMID:  11124949     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
K Hitomi; H Nakamura; S T Kim; T Mizukoshi; T Ishikawa; S Iwai; T Todo
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2000-12-21
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  276     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2001 Mar 
Date Detail:
Created Date:  2001-03-27     Completed Date:  2001-05-10     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  10103-9     Citation Subset:  IM    
Affiliation:
Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.
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MeSH Terms
Descriptor/Qualifier:
Alanine / chemistry
Amino Acid Sequence
Animals
Binding Sites
DNA Repair
Deoxyribodipyrimidine Photo-Lyase / chemistry*,  genetics*,  metabolism
Deuterium Oxide / chemistry
Glutamine / chemistry
Histidine / chemistry,  physiology*
Hydrogen-Ion Concentration
Leucine / chemistry
Models, Chemical
Models, Molecular
Molecular Sequence Data
Mutation
Protein Binding
Protons
Sequence Homology, Amino Acid
Substrate Specificity
Time Factors
Tryptophan / chemistry
Xenopus
Chemical
Reg. No./Substance:
0/Protons; 56-41-7/Alanine; 56-85-9/Glutamine; 61-90-5/Leucine; 71-00-1/Histidine; 73-22-3/Tryptophan; 7789-20-0/Deuterium Oxide; EC 4.1.99.3/Deoxyribodipyrimidine Photo-Lyase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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