title: Transition state in DNA polymerase β catalysis: rate-limiting chemistry altered by base-pair configuration.
abstract: Kinetics studies of dNTP analogues having pyrophosphate-mimicking β,γ-pCXYp leaving groups with variable X and Y substitution reveal striking differences in the chemical transition-state energy for DNA polymerase β that depend on all aspects of base-pairing configurations, including whether the incoming dNTP is a purine or pyrimidine and if base-pairings are right (T•A and G•C) or wrong (T•G and G•T). Brønsted plots of the catalytic rate constant (log(kpol)) versus pKa4 for the leaving group exhibit linear free energy relationships (LFERs) with negative slopes ranging from -0.6 to -2.0, consistent with chemical rate-determining transition-states in which the active-site adjusts to charge-stabilization demand during chemistry depending on base-pair configuration. The Brønsted slopes as well as the intercepts differ dramatically and provide the first direct evidence that dNTP base recognition by the enzyme-primer-template complex triggers a conformational change in the catalytic region of the active-site that significantly modifies the rate-determining chemical step.
authors.affiliation: Department of Biological Sciences and ‡Department of Chemistry, Dana and David Dornsife College of Letters, Arts and Sciences, University of Southern California , University Park Campus, Los Angeles, California 90089-0744, United States.
authors.forename: Keriann
authors.initials: K
authors.lastname: Oertell
authors.forename: Brian T
authors.initials: BT
authors.lastname: Chamberlain
authors.forename: Yue
authors.initials: Y
authors.lastname: Wu
authors.forename: Elena
authors.initials: E
authors.lastname: Ferri
authors.forename: Boris A
authors.initials: BA
authors.lastname: Kashemirov
authors.forename: William A
authors.initials: WA
authors.lastname: Beard
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
authors.forename: Charles E
authors.initials: CE
authors.lastname: McKenna
authors.forename: Myron F
authors.initials: MF
authors.lastname: Goodman
mesh_terms: D020029:Base Pairing / Q000235:genetics; D002384:Catalysis; D004249:DNA Damage / Q000235:genetics; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D011487:Protein Conformation
chemical_list: D019951:DNA Polymerase beta