title: Arginine as a general acid catalyst in serine recombinase-mediated DNA cleavage.
abstract: Members of the serine family of site-specific DNA recombinases use an unusual constellation of amino acids to catalyze the formation and resolution of a covalent protein-DNA intermediate. A recent high resolution structure of the catalytic domain of Sin, a particularly well characterized family member, provided a detailed view of the catalytic site. To determine how the enzyme might protonate and stabilize the 3'O leaving group in the strand cleavage reaction, we examined how replacing this oxygen with a sulfur affected the cleavage rate by WT and mutant enzymes. To facilitate direct comparison of the cleavage rates, key experiments used suicide substrates that prevented religation after cleavage. The catalytic defect associated with mutation of one of six highly conserved arginine residues, Arg-69 in Sin, was partially rescued by a 3' phosphorothiolate substrate. We conclude that Arg-69 has an important role in stabilizing the 3'O leaving group and is the prime candidate for the general acid that protonates the 3'O, in good agreement with the position it occupies in the high resolution structure of the active site of Sin.
authors: From the Department of Biochemistry and Molecular Biology and.
authors: Ross A
authors: RA
authors: Keenholtz
authors: Kent W
authors: KW
authors: Mouw
authors: Martin R
authors: MR
authors: Boocock
authors: Nan-Sheng
authors: NS
authors: Li
authors: Joseph A
authors: JA
authors: Piccirilli
authors: Phoebe A
authors: PA
authors: Rice
mesh_terms: D000143:Acids / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D053837:DNA Cleavage; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation; D045522:Recombinases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000143:Acids; D050505:Mutant Proteins; D045522:Recombinases; D012694:Serine; D001120:Arginine
keywords: Arginine; DNA Recombination; Enzyme Catalysis; Enzyme Mechanisms; General Acid; Nucleic Acid Enzymology; Phosphotransfer; Protein-DNA Interaction; Serine Recombinase; Site-specific Recombination
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