Kinetics of substrate recognition and cleavage by human 8-oxoguanine-DNA glycosylase.
Human 8-oxoguanine-DNA glycosylase (hOgg1) excises 8-oxo-7,8-dihydroguanine (8-oxoG) from damaged DNA. We report a pre-steady-state kinetic analysis of hOgg1 mechanism using stopped-flow and enzyme fluorescence monitoring. The kinetic scheme for hOgg1 processing an 8-oxoG:C-containing substrate was found to include at least three fast equilibrium steps followed by two slow, irreversible steps and another equilibrium step. The second irreversible step was rate-limiting overall. By comparing data from Ogg1 intrinsic fluorescence traces and from accumulation of products of different types, the irreversible steps were attributed to two main chemical steps of the Ogg1-catalyzed reaction: cleavage of the N-glycosidic bond of the damaged nucleotide and beta-elimination of its 3'-phosphate. The fast equilibrium steps were attributed to enzyme conformational changes during the recognition of 8-oxoG, and the final equilibrium, to binding of the reaction product by the enzyme. hOgg1 interacted with a substrate containing an aldehydic AP site very slowly, but the addition of 8-bromoguanine (8-BrG) greatly accelerated the reaction, which was best described by two initial equilibrium steps followed by one irreversible chemical step and a final product release equilibrium step. The irreversible step may correspond to beta-elimination since it is the very step facilitated by 8-BrG.
Kuznetsov
Nikita A
NA
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences Novosibirsk 630090, Russia.
Koval
Vladimir V
VV
Zharkov
Dmitry O
DO
Nevinsky
Georgy A
GA
Douglas
Kenneth T
KT
Fedorova
Olga S
OS
D004247:DNA / Q000737:chemistry / Q000378:metabolism; D045647:DNA Glycosylases / Q000737:chemistry / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism; D043382:DNA-Formamidopyrimidine Glycosylase / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005453:Fluorescence; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D013379:Substrate Specificity
C502319:8-bromoguanine; D029968:Escherichia coli Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase