The base-excision repair (BER) glycosylase, MutY, catalyzes the removal of adenine (A) mispaired across from 8-oxo-7,8-dihydroguanosine (OG). The latest mechanism reported for Geobacillus stearothermophilus (Gs) MutY is guided, first, by observations of the X-ray crystal structure obtained in our laboratory of the enzyme bound to an oligonucleotide containing the 1N transition-state analogue (TSA). Secondly, methanolysis experiments performed in our laboratory have determined Gs MutY to be a retaining glycosylase. A notable feature of this updated mechanism is the proposal that the catalytic Glu of Gs MutY (E43) acts as both a general acid and base as a means to protonate N7 of adenine for the initiation of glycosidic bond cleavage and to activate a nucleophilic water molecule for the formation of the abasic site, respectively. However, until this work, there was little understanding on the details of how E43 is able to adopt both protonation states over the course of catalysis. Here, we report new work on the Gs MutY ortholog which illustrates a network of charge-charge interactions with E43, suggesting the idea of pKa cycling. Kinetics characterization of the newly prepared Y126G Gs MutY variant reiterates the importance of the Y126 residue for catalysis. This result is supplemented by crystal structures of Y126F/S Gs MutY variants bound to oligonucleotides containing the 1N TSA. Together, these results suggest the importance of Y126 as a hydrogen bond donor to E43 and as a modulator of the solvation of the active site. pH-dependent kinetic characterization of WT Gs MutY and the Y126G variant further suggests the modulation of the pKa of E43 through its hydrogen bond contact with Y126. When PROPKA was utilized to computationally confirm these results, an unexpected Coulombic interaction between E43 and E188 was predicted. Interestingly, pH-dependent kinetic characterization of the E188A variant suggested that the titration of the E188 residue is also catalytically important. Altogether, the charge-charge interactions observed between E43 to both Y126 and E188 illustrate a mechanistic picture of pKa cycling for the catalytic Glu.

X-ray crystallography, site-directed mutagenesis, and computational chemistry illustrates a pKa cycling mechanism for MutY

ACS Fall 2021

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