Investigation of the role of the histidine-aspartate pair in the human exonuclease III-like abasic endonuclease, Ape1.
Hydrogen bonded histidine-aspartate (His-Asp) pairs are critical constituents in several key enzymatic reactions. To date, the role that these pairs play in catalysis is best understood in serine and trypsin-like proteases, where structural and biochemical NMR studies have revealed important pK(a) values and hydrogen bonding patterns within the catalytic pocket. However, the role of the His-Asp pair in metal-assisted catalysis is less clear. Here, we apply liquid-state NMR to investigate the role of a critical histidine residue of apurinic endonuclease 1 (Ape1), a human DNA repair enzyme that cleaves adjacent to abasic sites in DNA using one or more divalent cations and an active-site His-Asp pair. The results of these studies suggest that the Ape1 His-Asp pair does not function as either a general base catalyst or a metal ligand. Rather, the pair likely stabilizes the pentavalent transition state necessary for phospho-transfer.
Lowry
David F
DF
Macromolecular Structure & Dynamics, Environmental Molecular Sciences Laboratory, Richland, WA 99352, USA. david.lowry@pnl.gov
Hoyt
David W
DW
Khazi
Fayaz A
FA
Bagu
John
J
Lindsey
Andrea G
AG
Wilson
David M
DM
D001224:Aspartic Acid / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D019757:Carbon-Oxygen Lyases / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010165:Palladium / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D001224:Aspartic Acid; D006639:Histidine; D010165:Palladium; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008274:Magnesium; D002118:Calcium