Theoretical and mass spectrometric studies of damaged nucleobases and analogs toward understanding glycosylase mechanisms

The focus of this thesis is the examination of the thermochemical properties, primarily the gas phase acidity, proton affinity, and leaving group (LG) ability of damaged nucleobases and related species via mass spectrometry (FT-ICR, ion trap) and theoretical studies (quantum mechanical calculations). Our main hypothesis is that the study of intrinsic, gas-phase properties of the damaged nucleobases will lend insight into the mechanism of their excision from DNA. We study damaged nucleobases and analogs that are cleaved from DNA by various glycosylases: uracil-DNA glycosylase (UDG), 3-methyladenine glycosylase II (AlkA), and MutY glycosylase.;The LG ability of the N1-deprotonated 3-methyluracil anion relative to the N1-deprotonated 3-methylthymine anion is examined in the context of the UDG enzymatic reaction that excises uracil but not thymine from DNA. We confirmed that despite the close acidities uracil is a much better LG in the gas phase. Another interesting disparity between the LG ability and acidity is discovered for uracil substrates: when we examined hydrochloric acid and 3-methyluracil in the gas phase we found that despite similar acidities, chloride is a better LG than N1-deprotonated 3-methyluracil. We propose that the difference in LG ability is due to the different natures of the LGs (resonance vs. inductive stabilization). To test the hypothesis, a series of pyridone substrates were designed and examined.;AlkA is an enzyme that cleaves a wide range of damaged bases from DNA. Herein we examine 3- and 7-methylated AlkA purine substrates. The damaged nucleobases are found to be more acidic than the normal nucleobases. Because of this increased acidity, the damaged bases would be expected to be more easily cleaved from DNA by AlkA (their conjugate bases should be better LGs). We find that the acidity correlates to the AlkA excision rates, which lends support to an AlkA mechanism wherein the enzyme provides a nonspecific active site, and nucleobase cleavage is dependent on the intrinsic N-glycosidic bond stability.;The acidities and proton affinities of adenine and six adenine analogs that were designed to test various features of the enzyme MutY are also studied to allow better understanding of the mechanism of adenine removal by MutY.