AroA catalyzes the sixth step of the shikimate biosynthetic pathway which produces aromatiG amino acids in plants and bacteria, but is absent in mammals. This makes AroA an attractive antimicrobial target. The transition state (TS) structures of AroA- and acid-catalyzed 5-eno/pyruvyl shikimate-3-phosphate (EPSP) hydrolysis were studied in atomic detail by kinetic isotope effect (KIE) measurement. Enzymes bind their transition states more tightly than any other species, so molecules that closely resemble the transition state would have a high affinity for the enzyme and be good inhibitors. Radiolabelled EPSPs were synthesized and a KIE measurement method was developed. Six KIEs were measured for both the AroA- and acid-catalyzed reactions. KIEs for the AroA reaction indicate a cationic TS structure. The acid-catalyzed reaction may employ a slightly different mechanism with an earlier TS. A computational TS model was found and its KIEs were calculated. It demonstrated good agreement with the experimental values at most positions. The model is being modified to improve the agreement with the experimental KIEs. This TS structure will be a good starting point for inhibitor design. All these efforts, hopefully, can make a positive contribution to the development of antimicrobial drugs. / Thesis / Master of Science (MSc)

<p> AlkA is a monofunctional DNA glycosylase from E.coli. This enzyme catalyzes the hydrolysis of the N-glycosidic bond, initiating the first step in the base excision repair pathway. This activity is crucial to the maintenance of the genetic code, as the persistence of DNA aberrations can have significant cellular consequences including mutation, and inhibition of DNA replication and transcription. This enzyme has a broad substrate specificity catalyzing the excision of various lesions (including alkylation, oxidation and deamination products) from DNA. While biochemical and structural studies have been carried out on AlkA; how this enzyme is able to recognize and excise a variety of structurally diverse lesions from DNA and the mechanism by which this excision occurs remains unknown. In this study we have shown that a stem-loop DNA structure containing a hypoxanthine bulge is an optimal substrate for TS analysis of AlkA-catalyzed N-glycoside hydrolysis. In addition, we have developed methods to synthesize radiolabeled deoxyinosine triphosphate (diTP) and incorporate this radiolabeled nucleotide into the stem-loop DNA structure. We have developed a facile method of purification for his-tagged AlkA and his-tagged AlkA containing a TEV protease recognition site (for removal of the his-tag), and have shown that these proteins display an activity similar to that of wild-type AlkA. The [1'-3H] KIE was measured using liquid scintillation in a proof-of-principle experiment. The observed value of 1.046 is indicative of either a relatively synchronous ANDN (SN2) TS or an early DN*AN (SN1) TS with oxacarbenium ion character in the sugar ring, but significant bond order to the leaving group base still remaining. Future work involves repeat measurements of the [1 '-3H] KIE to validate the accuracy of the measurement observed here, examination of commitment to catalysis and optimization of the hypoxanthine bulge substrate synthesis. Analysis of KIEs at additional sites on the hypoxanthine base and sugar ring will contribute to TS analysis of AlkA-catalyzed N-glycoside hydrolysis and help elucidate the mechanism of hydrolysis. </p> / Thesis / Master of Science (MSc)

Formaldehyde emissions from non-structural wood composites are regulated and the regulation target is urea-formaldehyde (UF) resin. UF resins are hydrolytically unstable and constantly emit formaldehyde as a function of temperature and relative humidity. When heated, wood also generates formaldehyde, but this was of little concern until 2010 when formaldehyde regulations became much more demanding. This regulation motivated the industry to account for all formaldehyde sources, synthetic as from resin, and biogenic as from wood. This effort represents first steps towards quantifying biogenic and synthetic contributions to formaldehyde emissions in non-structural wood composites. It is possible to distinguish the 13C/12C isotope ratio of UF resins from the isotope ratio of plant biomass. Conditions during and after composite hot-pressing promote reactions that generate formaldehyde from wood and UF resin, and the kinetic isotope effect continuously lowers the product isotope ratios as a function of yield. If such isotope fractionation did not occur, it would be a simple matter to quantify contributions of wood and UF resin to formaldehyde emissions using static isotope ratios. Isotope fractionation, therefore, complicates the requirements for distinguishing biogenic and synthetic formaldehyde in wood composite emissions. Those requirements are 1) establish the reference carbon isotope ratios in wood and in UF resin (just the formaldehyde portion of UF), and 2) estimate the kinetic isotope effects in formaldehyde generation by wood and cured UF resin. The latter requirement fixes a range for the respective isotope ratios; the numerical ranges enable a simple model of the average isotope ratio for a mixture of biogenic and synthetic formaldehyde in wood composite emissions. Finally, the measured isotope ratio of captured emissions would be compared to the model. This work did not achieve all aspects of the requirements mentioned, but a solid foundation was established for future completion of the ultimate goals. In reference to requirement 1, the carbon isotope ratio of experimental Pinus taeda wood was accurately measured (including some isolated fractions) using isotope ratio mass spectroscopy (IRMS). IRMS of UF resin first requires removal of urea carbons- UF resin was subjected to acid hydrolysis and capture of the resin formaldehyde into aqueous ammonium hydroxide. This provided a nearly quantitative conversion (negligible isotope fractionation) of resin formaldehyde into hexamine for IRMS. Using this hexamine method, the formaldehyde carbon isotope ratios of two industrial UF resins were accurately measured, demonstrating basic feasibility for the project goal. Estimating the kinetic isotope effect (Requirement 2) required creation of a thermochemical reactor, where wood or cured UF resin was heated under N2 flow such that the emitted formaldehyde was easily captured. In this case, conversion of captured formaldehyde into hexamine was abandoned in favor of silica gel cartridges loaded with sodium bisulfite. Isolation and IRMS of the formaldehyde-bisulfite adduct were effective and considered easily transferable to industrial settings. This system was employed to measure fractionation in cured resin as a function of relative humidity, and in Pinus taeda wood as a function of relative humidity, temperature, and time. More information about isotope fractionation is required; but most notable was the fractionation behavior in wood where evidence was found for multiple formaldehyde generating reactions. Overall, this work established feasibility for the goals and laid the foundation for future efforts. / Master of Science / Home-interior products like cabinetry are often produced with wood composites adhesively bonded with urea-formaldehyde (UF) resin. UF resins are low cost and highly effective, but their chemical nature results in formaldehyde emission from the composite. High emissions are avoided, and the federal government has regulated and steadily reduced allowable emissions since 1985. The industry continuously improved UF technologies to meet regulations, as in 2010 when the most demanding regulations were implemented. At that time, many people were unaware that wood also generates formaldehyde; this occurs at very low levels but heating during composite manufacture causes a temporary burst of natural formaldehyde. Some wood types produce unusually high formaldehyde levels, making regulation compliance more difficult. This situation, and the desire to raise public awareness, created a major industrial goal: determine how much formaldehyde emission originates from the resin and how much originates from the wood. These formaldehyde sources can be distinguished by measuring the carbon isotope ratio, 13C/12C. This ratio changes and varies due to the kinetic isotope effect. Slight differences in 13C and 12C reactivity reveal the source as either petrochemical (synthetic formaldehyde) or plant-based (biogenic formaldehyde). This work demonstrates that achieving the industry goal is entirely feasible, and it provides the analytical foundation. The technical strategy is: 1) establish reference isotope ratios in wood and in UF resin, and 2) from the corresponding wood composite, capture formaldehyde emissions, measure the isotope ratio, and simply calculate the percentage contributions from the reference sources. However, a complication exists. When the reference sources generate formaldehyde, the respective isotope ratios change systematically in a process called isotope fractionation (another term for the kinetic isotope effect). Consequently, this effort developed methods to measure fractionation when cured UF resin and wood separately generate formaldehyde, with greater emphasis on wood. Isotope fractionation in wood revealed multiple fractionation mechanisms. This complexity presents intriguing possibilities for new perspectives on formaldehyde emission from wood and cured UF resin. In summary, this work demonstrated how source contributions to formaldehyde emissions can be determined; it established effective methods required to refine and perfect the approach, and it revealed that isotope fractionation could serve as an entirely novel tool in the materials science of wood composites.

How the fast (femtosecond-picosecond, fs-ps) protein dynamics contribute to enzymatic function has gained popularity in modern enzymology. With multiple experimental and theoretical studies developed, the most challenging part is to assess both the chemical step kinetics and the relevant motions at the transition state (TS) on the fast time scale. Formate dehydrogenase (FDH), which catalyzes a single hydride transfer reaction, is a model system to address this specific issue. I have crystallized and solved the structure of FDH from Candida boidinii (CbFDH) in complex with NAD+ and azide. With the guidance of the structure information, two active site residues were identified, V123 and I175, which could be responsible for the narrow donor-acceptor-distance (DAD) distribution observed in the wild type CbFDH. This thesis describes studies using kinetic isotope effects (KIEs) and their temperature dependence together with two-dimensional infrared spectroscopy on the recombinant CbFDH and its V123 and I175 mutants. Those mutants were designed to systematically reduce the size of their side chain (I175V, I175A, V123A, V123G and double mutant I175V/V123A), leading to broader distribution of DADs. The kinetic experiments identified a correlation between the DAD distribution and the intrinsic KIEs. The contribution of the fs-ps dynamics was examined via two-dimensional infrared spectroscopy (2D IR) by measuring the vibrational relaxation of TS analog inhibitor, aizde, reflecting the TS environmental motions. Our results provide a test of models for the kinetics of the enzyme-catalyzed reaction that invokes motions of the enzyme at the fs-ps time scale to explain the temperature dependence of intrinsic KIEs.

The relationship between protein structure, motions, and catalytic activity is an evolving perspective in enzymology. An interactive approach, where experimental and theoretical studies examine the same catalytic mechanism, is instrumental in addressing this issue. We combine various techniques, including steady state and pre-steady state kinetics, temperature dependence of kinetic isotope effects (KIEs), site-directed mutagenesis, X-ray crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations, to study the catalytic mechanisms of thymidylate synthase (TSase). Since TSase catalyzes the last step of the sole intracellular de novo synthesis of thymidylate (i.e. the DNA base T), it is a common target for antibiotic and anticancer drugs. The proposed catalytic mechanism for TSase comprises a series of bond cleavages and formations including activation of two C-H bonds: a rate-limiting C-H→C hydride transfer and a faster C-H→O proton transfer. This provides an excellent model system to examine the structural and dynamic effects of the enzyme on different C-H cleavage steps in the same catalyzed reaction. Our experiments found that the KIE on the hydride transfer is temperature independent while the KIE on the proton transfer is temperature dependent, implying the protein environment is better organized for H-tunneling in the former. Our QM/MM calculations revealed that the hydride transfer has a transition state (TS) that is invariable with temperature while the proton transfer has multiple subsets of TS structures, which corroborates with our experimental results. The calculations also suggest that collective protein motions rearrange the network of H-bonds to accompany structural changes in the ligands during and between chemical transformations. These computational results not only illustrate functionalities of specific protein residues that reconcile many previous experimental observations, but also provide guidance for future experiments to verify the proposed mechanisms. In addition, we conducted experiments to examine the importance of long-range interactions in TSase-catalyzed reaction, using both kinetic and structural analysis. Those experiments found that a remote mutation affects the hydride transfer by disrupting concerted protein motions, and Mg2+ binds to the surface of TSase and affects the hydride transfer at the interior active site. Both our experiments and computations have exposed interesting features of ecTSase that can potentially provide new targets for antibiotic drugs targeting DNA biosynthesis. The relationship between protein structure, motions, and catalytic activity learned from this project may have general implications to the question of how enzymes work.

Protein motions are complex, including occurring at different time scales, and their roles in enzyme-catalyzed reactions have always been of great interest among enzymologists. In order to characterize the potential factors that play a role on the chemical step of enzymatic reactions, variants of dihydrofolate reductase have been used as a benchmark system to study the motions of proteins correlated with the chemical step. A “global dynamic network” of coupled residues in Escherichia coli dihydrofolate reductase (ecDHFR), which assists in catalyzing the chemical step, has been demonstrated through quantum mechanical/molecular mechanical and molecular dynamic (QM/MM/MD) simulations, as well as bioinformatic analyses. A few specific residues — M42, G121, and I14 — were shown to function synergistically with measurements of single turnover rates and the temperature dependence of intrinsic kinetic isotope effects (KIEsint) of site-directed mutants. Although similar networks have been found in other enzymes, the general features of these networks are still unclear. This project focuses on exploring homologous residues of the proposed global network in human DHFR through computer simulations and measurements of the temperature dependence of KIEsint. The mutants M53W and S145V, both remote residues, showed significant decreases in catalytic efficiency. Non-additive isotope effects on activation energy were observed between M53 and S145, indicating their synergistic effect on hydride transfer in human DHFR. Apart from the effects of the conserved residues, we also extend our studies to exploring three potential phylogenetic events that account for the discrepancies between E. coli and human DHFR. They are L28, PP insertion and PEKN insertions by phylogenetic sequence analysis. Two of them (N23PP and G51PEKN E. coli DHFR) have been proved to be important both by MD simulation and experimental probe of KIEs measurement. The experiments have found that PP insertion itself rigidified the M20 loop and motions coupled to hydride transfer were impaired, however, loop rigidification was improved after incorporating PEKN. Furthermore, deletion of PP and PEKN of the engineered human enzyme also show a similar outcome. However, the effect of the key residue of L28 is not clear. In this project, we have step-wise engineered the human DHFR to be like hagfish (F31M) and E. coli (F32L). And it is found out that there is an increase in the temperature dependence of KIEs when the enzyme was bacterilized into a more primitive variant. This indicates that not only is residue F32 important and correlated with the chemical step as indicated by bioinformatic studies, but it is possible to trace the evolutionary trajectory. A triple mutation F32L-PP26N-PEKN62G on the human DHFR was also conducted, and it is not surprising to find out that the temperature dependence of KIEs has retained its behavior like wild-type human DHFR. These results suggest that the three predicted phylogenetically coherent events coevolved together to maintain the evolutionary preservation of the protein dynamics to enable H-tunneling from well-reorganized active sites. As has been indicated by the previous project, as the enzyme evolves, the active site of the enzyme will “reorganize” to form the optimal transition state for chemical step (from F32L-F32M-wild type DHFR). Here in this project, we aimed to systematically address this point of view through a series of cyclic permutation DHFR from directed evolutions. As this primitive enzyme is 7 orders of magnitude less efficient than the well-evolved human DHFR, together with four generations of evolved variants (cp, cp’ and cp”), this provides a good model system for explorations of the molecular basis of enzyme evolution. It is found that the organizations of transition state are improved before the catalytic efficiency is enhanced as the enzyme evolves.

The present dissertation seeks to deepen our understanding of hydrogen transfers and especially C-H bond activations in enzymes. Hydrogen transfers are ubiquitous in chemistry and biology and a thorough understanding of how they occur and what factors influence them will facilitate developments in biomimetic catalysis, rational drug design, and other fields. A particular difficulty with H-transfers is the importance of nuclear quantum effects to the reaction, particularly tunneling. The overall scope of the work here aims to examine how experimental kinetic isotope effects (KIEs) can be interpreted with a particular type of tunneling model, referred to as Marcus-like models, to yield a semi-quantitative picture of the physical mechanisms of H-transfers. Previous work had used this kind of model to qualitatively interpret experimental data using a combination of intuition and generalized theories. The work here examines these theories in quantitative detail, testing and calibrating our intuition in the context of several experimental systems. The first chapter of research (ch. II) focusses on the temperature dependence of primary KIEs and how these experiments can be quantitatively interpreted as a probe for certain kinds of enzyme or solvent dynamics. The subsequent chapters (ch. III-VI) focus on the use of secondary KIEs to determine the detailed structures of tunneling ready states (TRSs) and how the dynamics of H-tunneling affect those structures. These chapters focus primarily on the TRS of the enzyme alcohol dehydrogenase, but by examining an uncatalyzed analogue to that reaction (ch. VI), the work gains some insight about similarities and differences between catalyzed and uncatalyzed reactions. In summary, the work uncovers some principles of catalysis, not just the mechanism of a catalyzed reaction. The mechanism of C-H activation presented here provides an elegant solution to problems that have been vexing to accommodate within traditional models. This work constitutes some initial steps in making Marcus-like models quantitatively useful as a supplement or even replacement for traditional models of reactivity.

Protein-tyrosine phosphatases (PTPs) catalyze the hydrolysis of phosphorylated tyrosines by a 2-step mechanism involving nucleophilic attack by cysteine and general acid catalysis by aspartic acid. In most PTPs the aspartic acid resides on a flexible protein loop, consisting of about a dozen residues, called the WPD loop. PTP catalysis rates span several orders of magnitude, and differences in WPD loop dynamics have recently been show to correlate with the rate of enzymatic catalysis. The rate of WPD loop motion could possibly be related to a widely conserved tryptophan residue on the WPD loop. Therefore, point mutants were made in PTP1B (a human PTP) to the conserved tryptophan residue and their effects on catalytic rate and chemical reaction were studied. The results of these studies are presented in this thesis.

Choline oxidase (E.C. 1.1.3.17) from Arthrobacter globiformis catalyzes the four-electron oxidation of choline to glycine betaine (N,N,N-trimethylglycine) via two sequential, FAD-dependent reactions in which betaine aldehyde is formed as an enzyme-bound intermediate. In each oxidative half-reaction, molecular oxygen acts as electron acceptor and is converted into hydrogen peroxide. Biochemical, structural, and mechanistic studies on the wild-type and a number of mutant variants of choline oxidase have recently been carried out, allowing for the depiction of the mechanism of alcohol oxidation catalyzed by the enzyme. Catalysis by choline oxidase is initiated by the removal of the hydroxyl proton of alcohol substrate by a catalytic base in the enzyme-substrate complex, yielding the formation of the alkoxide species. In this dissertation, the roles of His351 and conserved His466 were investigated. The results presented demonstrate that His351 is involved in the stabilization of the transition state for the hydride transfer reaction and contributes to substrate binding. His466 is likely to be a catalytic base in choline oxidase due to its dramatic effect on enzymatic activity. Comparison of choline oxidase and other enzymes within its superfamily reveals the presence of a conserved His-Asn pair within the active site of enzymes. Therefore, the role of the conserved Asn510 in choline oxidase was examined in this study. The results presented here establish the importance of Asn510 in both the reductive and oxidative half-reactions. The lost of ability to form a hydrogen bond interaction between the side chain at position 510 with neighboring residues such as His466 resulted in a change from stepwise to concerted mechanism for the cleavages of OH and CH bonds of choline, as seen in the Asn510Ala mutant. Finally, the steady-state kinetic mechanism of pyranose 2-oxidase in the pH range from 5.5 to 8.5 was investigated. It was found that pH exerts significant effects on enzyme mechanism. This study has established the involvement of the residues in the initiation of enzyme catalysis and the stabilization of the alkoxide intermediate in choline oxidase. In addition, this work demonstrates the first instance in which the kinetic mechanism of a flavin-dependent oxidase is governed by pH.

Mechanistic studies of electrochemical proton coupled electron transfer (PCET) have attracted attention for many decades due to their importance in many fields ranging from electrocatalysis to biology. However, mechanistic research is confined to only a few groups, and challenges in this field can be found in both theory and experiment. The contributions to mechanistic studies of electrochemical PCET reaction in this thesis can be categorized under the following two headings: 1) mechanistic studies of an aminobenzoquinone modified monolayer system with multiple electron/proton transfer reaction; 2) studies that attempt to develop the relationship between thermochemical data and electrochemical PCET mechanism. An aminobenzoquinone modified monolayer showing nearly ideal electrochemical behavior and high stability was successfully prepared and used as a model system for the mechanistic study of electrochemical multiple electron/proton transfer. This model system has been proposed to undergo a 2e3H transfer at low pH electrolyte and a 2e2H transfer at high pH electrolyte. Two non-destructive electrochemical techniques (cyclic voltammetry and chronocoulmetry) have been applied for the measurement of apparent standard rate constant as a function of pH. Both pH dependent apparent formal potential and pH dependent apparent standard rate constant have been used to determine the charge transfer mechanism of this monolayer system. Under the assumption of an operative PCET mechanism (i.e. electron transfer step is the rate determining step), a theoretical description of this system has been developed based on the refinement and extension of previous models. By combining this extended theoretical model with pH dependent apparent formal potential and apparent standard rate constant, charge transfer pathways have been determined and shown to be consistent with the observed pH dependent electrochemical response, in addition, the determined pathways in this aminobenzoquinone modified monolayer are similar to previous reported pathways for benzoquinone freely dissolved in aqueous buffered electrolyte. A series of analytical expressions built in this thesis demonstrate that the parameters that differentiate stepwise mechanisms from concerted mechanisms can be classified into two aspects: thermodynamic parameters, namely acid dissociation constants, standard formal potentials; and kinetic parameters, namely standard rate constants, standard transfer coefficients. Although attempts to understand the relation between controlling parameters and electrochemical PCET mechanism (stepwise versus concerted) has been reported previously by some groups, there are still lots of unresolved aspects requiring further investigation. In this thesis, an important conclusion has been drawn which is that for the stepwise mechanism, an apparent experimentally observable kinetic isotope effect (KIE) can be induced by solvent isotope induced variation of acid dissociation constants, which contradicts previous understanding. Additionally, for the first time, values of apparent KIE, which were measured for the aminobenzoquinone modified monolayer system with stepwise PCET mechanism, were successfully explained by variation in acid dissociation constants, not by variation in standard rate constants. Based on theoretical prediction, a nitroxyl radical modified bilayer showing one electron one proton transfer reaction has been prepared in an effort to afford experimental verification. After applying similar analytical procedures as those for the aminobenzoquinone modified monolayer system, this bilayer system has been shown to follow the concerted 1e1H transfer pathway in high pH electrolytes. These latter contributions provide evidence that further development in this field will eventually lead to a comprehensive theory that can use known thermochemical variables to fully predict PCET mechanism.