| The active site chemistry of the E. coli DNA repair adenine glycosylase MutY has been characterized by direct experimental comparisons with other base excision repair (BER) enzymes, and by targeted mutagenesis studies. This work has shown that MutY is a monofunctional glycosylase that does not cleave DNA concomitantly with base excision catalysis. However, it has been demonstrated that the MutY catalytic pathway involves a covalent intermediate characteristic of bifunctional BER glycosylase/AP lyases, which cause DNA strand cleavage at a rate equal to base excision.;The active site lysine at position 142 has been identified as the primary residue involved in forming the transient covalent intermediate during catalysis, bestowing MutY with this unusual behavior. Furthermore, the K142A MutY form showed kinetic behavior unchanged from that of wild-type MutY, indicating that the covalent intermediate present during catalysis is mechanistically decoupled from the process of base excision.;DNA strand cleaving AP lyase activity was successfully engineered into MutY by replacing serine 120 with lysine. Furthermore, it has been shown that lysine at position 120 is capable of forming covalent intermediates with DNA in addition to lysine 142. Based upon protein primary sequence alignments with structurally related BER enzyme superfamily members, it is apparent that a conserved lysine at positions analogous to position 120 in MutY is important for the presence of an associated glycosylase/AP lyase activity.;The replacement of two amino acid residues in MutY that are highly conserved in the BER enzyme superfamily results in a loss of catalytic activity, yet both replacement forms show an ability to bind substrate and substrate analogue DNA with high affinity. This tight association may prove useful in the future acquisition of crystallographic data regarding substrate DNA positioning in the active site of MutY, without the complications of product conversion.;The results reported herein represent a significant increase in the understanding of the active site chemistry of MutY. Since MutY belongs to a large group of BER enzymes sharing similar active site motifs, this information has given valuable insight regarding a large subset of enzymes involved in the important process of base excision DNA repair. |
