Abstract
The mechanism of the Escherichia coli DNA T:G mismatch endonuclease (Vsr) has been investigated using oligodeoxynucleotides substituted, at the scissile phosphate, with isomeric phosphorothioates and a 3'-phosphorothiolate. Binding and kinetic data with the phosphorothioates/phosphorothiolate indicate that the two magnesium ions, which constitute essential co-factors, are required to stabilise the extra negative charge developed on the phosphate as the transition state is formed. Additionally one of the magnesium ions serves to activate the leaving group (the non-bridging 3'-oxygen atom of the scissile phosphate) during the hydrolysis reaction. Stereochemical analysis, using the R(p) phosphorothioate isomer, indicates that Vsr carries out a hydrolytic reaction with inversion of stereochemistry at phosphorus, compatible with an in-line attack of water and a pentacovalent transition state with trigonal bipyramidal geometry. In conjunction with structures of Vsr bound to its products, these data allow the reconstruction of the enzyme-substrate complex and a comprehensive description of the hydrolysis mechanism.
Sign-in/Register to access full text options 
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.
