PNNL

Abstract

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 pKa 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.