Catalytic contributions of key residues in the adenine glycosylase MutY revealed by pH-dependent kinetics and cellular repair assays.

MutY prevent DNA mutations associated with 8-oxoguanine (OG) by catalyzing the removal of adenines opposite OG. pH dependence of the adenine glycosylase activity establish that Asp 138 of MutY must be deprotonated for maximal activity consistent with its role in stabilizing the oxacarbenium ion transition state in an S(N)1 mechanism. A cellular OG:A repair assay allowed further validation of the critical role of Asp 138. Conservative substitutions of the catalytic residues Asp 138 and Glu 37 resulted in enzymes with a range of activity that were used to correlate the efficiency of adenine excision with overall OG:A repair and suppression of DNA mutations in vivo. The results show that MutY variations that exhibit reduced mismatch affinity result in more dramatic reductions in cellular OG:A repair than those that only compromise adenine excision catalysis.