Gas phase studies of nucleobases using computation and mass spectrometry
Creators: name: Zhang, Lanxin
Publisher: name: No Publisher Supplied
Year: 2023

description: This dissertation focuses on studying the gas-phase intrinsic properties of nucleobases and investigating their corresponding enzyme mechanism using computational (Gaussian) and experimental (mass spectrometry) methods. Chapter 2 delves into the reaction mechanism of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) ribosylation reaction. Within this context, two hypotheses are formulated concerning the sequence of nucleophilic attack and proton transfer steps specific to human HGPRT. These proposed mechanisms are associated with the proton affinity (PA) or acidity of the substrates involved. To shed light on these hypotheses, an investigation into the gas-phase acidities and proton affinities of the nucleobases acting as substrates for human HGPRT was conducted using a combination of theoretical and experimental approaches. The findings highlight a correlation between the PA of the N9 site and the enzyme reaction rate constant (kcat). This observation supports one of the proposed mechanisms, suggesting that the initial step involves a nucleophilic attack between the substrate and phosphoribosyl pyrophosphate (PRPP), followed by the subsequent proton transfer.Hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT), the homologs of human HGPRT in parasites Plasmodium falciparum and its substrates are studied in Chapter 3. This enzyme holds a significant position in the quest to develop effective antimalarial agents. In order to gain valuable insights into the mechanism of Pf HG(X)PRT, a comparison between the gas-phase acidities and proton affinities of the substrates and the kinetic parameters of Pf HG(X)PRT has been carried out. This scrutiny is undertaken to establish potential correlations. Both computational and experimental techniques have been employed to scrutinize the gas-phase acidities and PAs of the Pf HG(X)PRT substrates. Furthermore, a calculated kinetic isotope effect (KIE) study that holds the potential to discern between the plausible mechanisms, is subsequently validated by an experimental KIE result, providing a robust foundation for further mechanistic insights.Chapter 4 delves into a comprehensive exploration of parasites HG(X)PRT inhibitors and non-substrates in the gas phase, aiming to uncover the significance of the N7 and N9 sites within these enzyme substrate analogs. In addition, an experimental assessment of the less acidic site of xanthine is conducted. This empirical evaluation serves the purpose of corroborating the acidity of 7-methylxanthine, thereby underscoring the indispensable role of experimental findings in validating the computational dataset. The results indicate that the N9 site PA demonstrates higher values for HG(X)PRT substrates in comparison to both non-substrates and inhibitors. Furthermore, the absence of nitrogen atom at positions 7 or 9 in the purine results in diminished reactivity among the analogs, suggesting that the presence of these nitrogens is pivotal for maintaining optimal reactivity and binding affinity. This observation further substantiates the mechanisms elucidated in Chapters 2 and 3, reinforcing their validity.In Chapter 5, a comprehensive investigation has been undertaken to analyze the intrinsic properties of alkylated nucleobases that serve as substrates for Escherichia coli DNA dioxygenase enzyme AlkB. This examination is conducted through a combination of computational methods and empirical experimentation. The E. coli AlkB enzyme functions as a reparative agent for damaged substrates, effectively reversing alkyl lesions and restoring them to their original nucleotide states. Specifically, E. coli AlkB targets N1 site methylated purines, N3 site methylated pyrimidines, and etheno-bridged adducts as its primary substrates. In contrast, the E. coli glycosylase AlkA, another significant enzyme in response to alkylation damage, focuses on distinct substrates divergent from those addressed by AlkB. The analysis of substrates’ gas-phase N9-H acidity mainly elucidates the distinctive roles played by AlkB and its counterpart enzyme AlkA in the intricate process of DNA repair.Chapter 6 presents an intricate computational study focused on four distinct etheno adducts: 1,N6-ethenoadenine, 3,N4-ethenocytosine, N2,3-ethenoguanine and 1,N2-ethenoguanine. These etheno adducts are investigated within the context of the E. coli AlkB dealkylation mechanism. AlkB’s proposed mechanism for repairing etheno adducts unfolds through a two-step protonation pathway, with the second protonation step as the rate-limiting step in previous studies. In this chapter, a distinct mechanism is proposed for each etheno substrate, intricately tailored to the substrate’s characteristics. Through this proposed mechanism, the proton affinity (PA) of the corresponding structure is computed, rooted in the intricacies of the mechanism itself. The correlation established between the proton affinities (PAs) and the substrates’ reactivity underscores the feasibility of the proposed AlkB etheno substrate repair mechanism, simultaneously shedding light on the possibility of the second protonation as the rate-determining step in the dealkylation process.In Chapter 7, a collaborative work with Dr. Sheila David (University of California, Davis) is presented. Nei-like 1 glycosylase (NEIL1) substrates are studied in gas-phase computationally. From the calculations, a more stable lactim tautomer (2OH vs 4OH) might be the biologically relevant structure in the reaction, and the proton affinity of N3 of the base lesion tracks with the substrate relative excision rate. These trends provide evidence to the hypothesis that tautomerization and N3 protonation contribute to NEIL1 excision catalysis of these pyrimidine base lesions. Distinct substrate lesions, such as 5-hydroxycytosine (5-OHC) and 5-guanidinohydantoin (Gh), are more efficiently removed by the edited NEIL1 isoform, consistent with inherent differences in tautomerization and proton affinities.