Substitution of Asp-210 in HAP1 (APE/Ref-1) eliminates endonuclease activity but stabilises substrate binding

HAP1, also known as APE/Ref-1, is the major apurinic/apyrimidinic (AP) endo nuclease in human cells. Previous structural studies have suggested a possi ble role for the Asp-210 residue of HAP1 in the enzymatic function of this enzyme, Here, we demonstrate that substitution of Asp-210 by Asn or Ala eli minates the AP endonuclease activity of HAP1, while substitution by Glu red uces specific activity similar to 500-fold, Nevertheless, these mutant prot eins still bind efficiently to oligonucleotides containing either AP sites or the chemically unrelated bulky p-benzoquinone (pBQ) derivatives of dC, d A and dG, all of which are substrates for HAP1, These results indicate that Asp-210 is required for catalysis, but not substrate recognition, consiste nt with enzyme kinetic data indicating that the HAP1-D210E protein has a 30 00-fold reduced K-cat for AP site cleavage, but an unchanged K-m. Through a nalysis of the binding of Asp-210 substitution mutants to oligonucleotides containing either an AP site or a pBQ adduct, we conclude that the absence of Asp-210 allows the formation of a stable HAP1-substrate complex that exi sts only transiently during the catalytic cycle of wild-type HAP1 protein, We interpret these data in the context of the structure of the HAP1 active site and the recently determined co-crystal structure of HAP1 bound to DNA substrates.