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.
Highlights
Sin resolvase is a site-specific DNA recombinase that catalyzes phosphotransfer without the use of divalent cations
Because sulfur more stabilizes a negative charge than oxygen, a 3Ј thiolate is a better leaving group than a 3Ј alkoxide; it may not require protonation during catalysis and is predicted to be a better leaving group in the absence of a general acid
The pKa of arginine in water is ϳ12, which is quite high for a general acid catalyst, but the environment of an enzyme active site can dramatically shift the pKa values of critical residues
Summary
Sin resolvase is a site-specific DNA recombinase that catalyzes phosphotransfer without the use of divalent cations. In contrast to the metal ion containing active sites, the tyrosine recombinases such as Flp and Cre, and the related topoisomerase Ib use a different mechanism, which involves a lysine general acid to protonate the leaving 5Ј oxygen and either a histidine or a water to deprotonate the nucleophilic tyrosine They contain conserved arginines that probably orient the scissile phosphate and stabilize the transition state (26 –28). We find that mutations of Arg-69, but not of other active site residues, were partially rescued by the modified substrate We conclude that this highly conserved arginine residue likely contributes to catalysis of the cleavage reaction by protonating the 3Ј oxygen leaving group
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
