Conformational transitions in human AP endonuclease 1 and its active site mutant during abasic site repair.
AP endonuclease 1 (APE1) is a crucial enzyme of the base excision repair pathway (BER) in human cells. APE1 recognizes apurinic/apyrimidinic (AP) sites and makes a nick in the phosphodiester backbone 5' to them. The conformational dynamics and presteady-state kinetics of wild-type APE1 and its active site mutant, Y171F-P173L-N174K, have been studied. To observe conformational transitions occurring in the APE1 molecule during the catalytic cycle, we detected intrinsic tryptophan fluorescence of the enzyme under single turnover conditions. DNA duplexes containing a natural AP site, its tetrahydrofuran analogue, or a 2'-deoxyguanosine residue in the same position were used as specific substrates or ligands. The stopped-flow experiments have revealed high flexibility of the APE1 molecule and the complexity of the catalytic process. The fluorescent traces indicate that wild-type APE1 undergoes at least four conformational transitions during the processing of abasic sites in DNA. In contrast, nonspecific interactions of APE1 with undamaged DNA can be described by a two-step kinetic scheme. Rate and equilibrium constants were extracted from the stopped-flow and fluorescence titration data for all substrates, ligands, and products. A replacement of three residues at the enzymatic active site including the replacement of tyrosine 171 with phenylalanine in the enzyme active site resulted in a 2 x 10(4)-fold decrease in the reaction rate and reduced binding affinity. Our data indicate the important role of conformational changes in APE1 for substrate recognition and catalysis.
Kanazhevskaya
Lyubov Yu
LY
Institute of Chemical Biology & Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
Koval
Vladimir V
VV
Zharkov
Dmitry O
DO
Strauss
Phyllis R
PR
Fedorova
Olga S
OS
D002384:Catalysis; D020134:Catalytic Domain; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D007700:Kinetics; D020125:Mutation, Missense; D011487:Protein Conformation; D013379:Substrate Specificity
D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase