MECHANISM OF ACTION OF BASE RELEASE BY ESCHERICHIA-COLI FPG PROTEIN -ROLE OF LYSINE-155 IN CATALYSIS

Fpg protein (formamidopyrimidine/8-oxoguanine DNA N-glycosylase) is a DNA repair enzyme that catalyzes the removal of oxidized purines, most notably the mutagenic 7-hydro-8-oxoguanine (8oxoGua) lesion, by an N- glycosylase action. Additionally, Fpg protein catalyzes beta and delta elimination reactions subsequent to removal of the base lesions, as w ell as the analogous chemistry at abasic sites (AP sites). In this rep ort, we show that of the two lysines that are conserved among the vari ous putative prokaryotic Fpg proteins, a site specific alteration in o ne of them (lysine 155 changed to alanine) displays meaningful changes in substrate activities. However, lysine 155 is not required for the postulated covalent enzyme-substrate imine intermediate as demonstrate d by trapping of the mutant protein-oligonucleotide complexes with cya nide or cyanoborohydride. The K155A mutant shows a decrease in activit y with the 8oxoGua-substrate of similar to 50-fold under both k(cat)/K -m and k(cat) conditions. This mutant also displays a similar reductio n in activity with an oligonucleotide substrate possessing a single 2' -deoxy-8-oxonebularine site. In contrast, activity for a site specific 7-methylformamidopyrimidine-modified oligonucleotide is reduced simil ar to 3-4-fold, a much more modest decrease in activity. Interestingly , there is a concomitant increase in AP lyase activity above wild-type for the K155A mutant (1.6-fold increase in k(cat), 32-fold increase i n k(cat)/K-m), demonstrating retention of functional beta and delta ly ase activities. Together these observations are readily accommodated b y a model requiring a direct interaction of lysine 155 with the Cg oxy gen of 8-oxopurines. Thus, conservation of this amino acid residue dur ing evolution appears to be essential for specific incision of the mut agenic 8oxoGua base lesion by Fpg protein.