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MutY excises adenine (A) from 8-oxo-guanine:adenine (OG:A) lesions in DNA to initiate base excision repair (BER) and thereby prevent mutations. A catalytic Glu, found at position 43 in the enzyme fromGeobacillus stearothermophilus(GsMutY), protonates the nucleobase at N⁷to labilize the N-glycosidic bond. The resulting oxocarbenium ion transition state is stabilized by a covalent DNA-enzyme intermediate and resolved by nucleophilic attack to yield thebeta-anomer abasic AP site product. The retaining SN1 mechanism for MutY posits deprotonation of the nucleophile by the catalytic Glu. Here we tested kinetic and structural consequences of Glu replacement and found that E43Q and E43S substitution variants were severely impaired, retained measurable activity, but engage the substrate nucleobase in ananticonformation, rotated by 180° from thesynconformation seen in previous substrate complexes. The enzyme-generated AP product is observed in itsalpha-anomer configuration for these Glu-replacement variants. Comparison with inverting adenine glycosylases that act on RNA or nucleosides shows that MutY's mechanism is uniquely reliant on one catalytic residue for both leaving group and nucleophile activation, a situation that may serve to ensure only rare adenines paired with OG are excised.