Circadian oscillators or clocks are a widespread, endogenous class of oscillatory mechanisms that control the ~24h temporal pattern of diverse organism functions. In cyanobacteria this mechanism is formed by three proteins, KaiA, KaiB and KaiC. KaiA is shown here to be a two domain protein that directly interacts with KaiC and enhances the KaiC autokinase activity. The amino-terminal domain of KaiA can be structurally categorized as a pseudo-receiver, a class of proteins used in signaling cascades and activated by direct protein??protein interactions. The carboxy-terminal domain interacts directly with KaiC, is sufficient to enhance the KaiC autokinase activity in a manner similar to full-length KaiA, and adopts a unique, all α-helical dimeric fold. The structure of this domain raises interesting probabilities regarding the mode of KaiA??KaiC interaction. The two KaiA domains are shown to directly interact with each other, which suggests a possible mechanism of signal transfer from the amino to carboxy-terminal domain. Hydrogen bonds are of paramount importance in nucleic acid structure and function. Here we show that changes in the width and anharmonicity of vibrational potential energy wells of hydrogen bonded groups can be measured in nucleic acids and can possibly be correlated to structural properties, such as length. Deuterium/protium fractionation factors, which are sensitive to the vibrational potential well width, were measured for the imino sites of thymidine residues involved in A:T base pairs or free in solution, and a correlation was established between decreasing fractionation factors and increasing imino proton chemical shift, δH3. Similarly, a correlation was observed between δH3and deuterium isotope effects (DIE) on chemical shift of thymidine carbon atoms. Combined these results indicate that as hydrogen-bond strength increases the vibrational potential wells of imino protons widen with a corresponding increase in anharmonicity. However, trans-hydrogen bond DIE on carbon chemical shifts of A:T base-paired adenosine residues do not correlate with those measured on thymidine residues. We propose that this lack of correlation is due to DIE dependence on base-pair geometry, which is not easily measured by traditional NMR experiments.

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.

The phenylalanine hydroxylase from Chromobacterium violaceum (CvPheH) is a non-heme iron monooxygenase that catalyzes the hydroxylation of phenylalanine. This study presents the use of kinetic isotope effects (KIE) as mechanistic probes to compare the reactivity of CvPheH and that of the eukaryotic aromatic amino acid hydroxylases. This study also describes the use of different spectroscopic and kinetic techniques to identify the hydroxylating intermediate for this enzyme and the assignment of the NMR backbone resonances of CvPheH. Kinetic isotope effects on aromatic and benzylic hydroxylation were used to establish that bacterial and eukaryotic phenylalanine hydroxylases have similar reactivity. The observed KIE on aromatic hydroxylation of 1.4 was shown to be a combination of an inverse isotope effect on the hydroxylation of the amino acid and a normal isotope effect on a subsequent step in the reaction. An isotope effect on benzylic hydroxylation of 10 was found for CvPheH. This result establishes the similar reactivity for CvPheH and the eukaryotic aromatic amino acid hydroxylases and suggests the involvement of a common hydroxylating intermediate. Kinetic isotope effects were used to study the hydroxylation of the aliphatic substrate cyclohexylalanine. The Dkcat value with [1,2,2,3,3,4,4,5,5,6,6-2H11]- cyclohexylalanine is unity with wild-type CvPheH, suggesting that chemistry is not ratelimiting with this substrate. The intramolecular isotope effect calculated using [1,2,3,4,5,6-2H6]-cyclohexylalanine yields a value of 14. This result is evidence for the involvement of a reactive iron species capable of abstracting a hydrogen atom from the aliphatic carbon in cyclohexylalanine. Analysis of the CvPheH reaction using freeze-quench Mössbauer spectroscopy allowed the detection of an Fe(IV) species in the first turnover of the enzyme. Chemical quench and stopped-flow spectrophotometric methods were used to establish the kinetic competency of the Fe(IV) intermediate as the hydroxylating species. The NMR amide backbone resonances in the HSQC spectrum of CvPheH were assigned to the corresponding amino acid residues using a suite of TROSY-based threedimensional triple resonance experiments. We were able to assign 224 residues out of the 278 assignable residues in CvPheH, this constitutes 81 percent of the assignable protein sequence.

Circadian oscillators or clocks are a widespread, endogenous class of oscillatory mechanisms that control the ~24h temporal pattern of diverse organism functions. In cyanobacteria this mechanism is formed by three proteins, KaiA, KaiB and KaiC. KaiA is shown here to be a two domain protein that directly interacts with KaiC and enhances the KaiC autokinase activity. The amino-terminal domain of KaiA can be structurally categorized as a pseudo-receiver, a class of proteins used in signaling cascades and activated by direct protein??protein interactions. The carboxy-terminal domain interacts directly with KaiC, is sufficient to enhance the KaiC autokinase activity in a manner similar to full-length KaiA, and adopts a unique, all α-helical dimeric fold. The structure of this domain raises interesting probabilities regarding the mode of KaiA??KaiC interaction. The two KaiA domains are shown to directly interact with each other, which suggests a possible mechanism of signal transfer from the amino to carboxy-terminal domain. Hydrogen bonds are of paramount importance in nucleic acid structure and function. Here we show that changes in the width and anharmonicity of vibrational potential energy wells of hydrogen bonded groups can be measured in nucleic acids and can possibly be correlated to structural properties, such as length. Deuterium/protium fractionation factors, which are sensitive to the vibrational potential well width, were measured for the imino sites of thymidine residues involved in A:T base pairs or free in solution, and a correlation was established between decreasing fractionation factors and increasing imino proton chemical shift, δH3. Similarly, a correlation was observed between δH3and deuterium isotope effects (DIE) on chemical shift of thymidine carbon atoms. Combined these results indicate that as hydrogen-bond strength increases the vibrational potential wells of imino protons widen with a corresponding increase in anharmonicity. However, trans-hydrogen bond DIE on carbon chemical shifts of A:T base-paired adenosine residues do not correlate with those measured on thymidine residues. We propose that this lack of correlation is due to DIE dependence on base-pair geometry, which is not easily measured by traditional NMR experiments.

Polarized Raman spectroscopy has been used to measure the deuterium isotope effect, (delta)pK = pKD2O – pKH2O, for the second ionization constant of sulfuric acid in the temperature range of 25 °C to 200 °C at saturation pressure. Results for pK in light water agree with the literature within ± 0.034 pK units at alltemperatures under study, confirming the reliability of the method. The ionization constant of deuterated bisulfate, DSO4-, differs significantly from previous literature results at elevated temperatures. This results in an almost constant (delta)pK ≈ 0.425 ± 0.076 over the temperature range under study. Differences in (delta)pK values between the literature and current results can be attributed to the effect of dissolved silica from cell components. The new results are consistent with (delta)pK models that treat the temperature dependence of (delta)pK by considering differences in the zero-point energy of hydrogen bonds in the hydrated product and reactant species. The phosphate hydrolysis equilibrium was measured between the temperatures of 5 °C and 80 °C and the borate/boric acid equilibrium between the temperatures of 25 °C and 200 °C. The high alkalinity and temperatures experienced by these two systems had a significant impact on the glass dissolution and equilibrium. / Raman spectroscopy was used to measure the small differences in ionization constants for weak acids/bases as a function of temperature. / University of Guelph, Atomic Energy of Canada Limited (AECL), Bruce Power, University Network of Excellence in Nuclear Engineering (UNENE), National Sciences and Engineering Research Council of Canada (NSERC), Natural Resources Canada, Ontario Power Generation (OPG), Canada Foundation for Innovation

La medición de efectos isotópicos es una herramienta importan­te en el estudio de las transformaciones químicas. El uso de efec­tos isotópicos de átomos ligeros como el deuterio es muy común e incluso aparece en muchos textos básicos de química. Lamen­tablemente, el uso de efectos isotópicos de átomos pesados no ha recibido la misma atención a pesar de su gran utilidad. Este manuscrito sirve como introducción a este tema importante. / The determination of isotope effects is an important tool in the study of chemical transformations. Very common in the liter­ature is the use of deuterium isotope effects, which is typically covered in many textbooks. Unfortunately, heavy atom isotope effects have not received the same attention despite its great rel­evance. This article will serve as an introduction to this very important topic.

The nucleotide 2'-deoxythymidine 5'-monophosphate (thymidylate, dTMP) is phosphorylated twice to become a substrate for DNA polymerases, which copy a cell’s genetic information in advance of cell division. The main route to dTMP is mediated by the enzyme thymidylate synthase (TSase) and goes through 2'-deoxyuridine 5'-monophosphate (dUMP); dUMP’s heterocyclic aromatic pyrimidine ring loses a proton from its C5 position and gains a methylene and a hydride from the other reactant, methylene tetrahydrofolate (MTHF). In general, intricate knowledge of an enzyme’s mechanism can yield insight that leads to the development of precision-targeted inhibitors tailored exactly to thymidylate synthase. In fact, even more careful targeting could be achievable: Although E. coli TSase has served as a model system, investigators have increasingly been directing their lines of inquiry toward human TSase. A general enzymatic catalytic cascade is complex, comprising substrate binding, the chemical steps and product release; typically, the product release step is rate-limiting. TSase, however, is partially rate-limited by the chemistry portion of the process. The enzymatic mechanism has been considered for decades, yet recently has undergone a reassessment. After substrate binding – for which there is strong evidence for preference to dUMP as the first ligand in the wild-type E. coli enzyme – the important events are methylene transfer from MTHF to dUMP, proton abstraction and hydride transfer. The last of these – hydride transfer – is irreversible and rate-limiting (to a large degree without Mg2+, and to a small but noticeable degree with Mg2+). The studies described here are aimed at three therapeutically relevant questions: (a) determining the extent of negative charge accumulation at the O4 position of the hydride transfer acceptor; (b) expanding knowledge of the differential properties of E. coli and human TSase; and (c) gaining insight into the molecular origin of the drug resistance seen in a clinically relevant human TSase mutant. The properties touched on in this work include steady-state kinetics; inhibition constants toward 5-fluoro dUMP, substrate binding sequence and the temperature dependency of intrinsic hydride transfer kinetic isotope effects (KIEs). Intrinsic KIEs are a specialized measurement that permits the investigator to examine a particular hydrogen transfer step in isolation; it is achieved by labeling the bond to hydrogen broken in the reaction with protium (1H, also written as H), deuterium (2H, also written as D) or tritium (3H, also written as T). The latter is radioactive. The reaction is conducted with a mixture of two hydrogen isotopes at a time, and the extent to which the heavier isotope is disfavored against reaction is assessed; this covers multiple steps. Heavier isotopes directly participating in a chemical step react slower both because of zero-point vibrational energies if a semi-classical view is taken and because of the mass-dependence of tunneling probabilities if a quantum-mechanical view is taken. Each of the two-way isotopic comparisons mentioned above furnishes an observed KIE for that competition between two isotopes. Mathematical combination of two isotopic comparisons cancels out the effect of isotopically insensitive steps and provides rich insight into the hydride transfer alone. The ultimate result is the ratio of rate constants for the isotopologues; this ratio’s magnitude and variation with temperature report on the compactness of the active site and its resistance to thermal fluctuation, respectively. Our results reveal a possible role for E. coli asparagine 177 (N177) in the hydride transfer transition state (TS) stabilization, as revealed by its disruption in the aspartate mutant, N177D. This disruption was found to be alleviated to a high extent when the substrate was changed to dCMP, consistent with the N177 stabilizing partial negative charge at the TS for hydride transfer. This has drug design implications. Our work on human TSase underscores slightly weaker substrate binding preference, insensitivity to Mg2+ and mild alteration of hydride transfer TS when compared with E. coli TSase. Finally, analysis of the Y33H mutant of human TSase – the affected residue being remote from the active site – indicated the drug resistance was because of a higher inhibition constant for 5F-dUMP and that the hydride transfer step is disrupted, with a wider variation among donor-acceptor distances (between the two carbons involved in the hydride transfer at the TS for that step). Other researchers’ crystallographic evidence reveals greater positional uncertainty for a set of active-site side chains in the E. coli equivalent mutant. In totality, the data available implicate enzyme motions as relevant to drug binding and to catalysis for human TSase. In summary, the research described herein enriches the understanding of several aspects of the behavior of multiple TSase variants – the overall performance as seen via steady-state kinetics; the pattern of substrate binding as seen with observed KIEs for the proton abstraction step; and the efficiency of active site preparation for hydride transfer as evidenced in the temperature dependency of intrinsic hydride transfer KIEs.

Dissociation constants for alternate dirmcleotide substrates and competitive inhibitors suggest that the dinucleotide binding site of the Ascaris suum NAD-malic enzyme is hydrophobic in the vicinity of the nicotinamide ring. Changes in the divalent metal ion activator from Mg^2+ to Mn^2+ or Cd^2+ results in a decrease in the dinucleotide affinity and an increase in the affinity for malate. Primary deuterium and 13-C isotope effects obtained with the different metal ions suggest either a change in the transition state structure for the hydride transfer or decarboxylation steps or both. Deuterium isotope effects are finite whether reactants are maintained at saturating or limiting concentrations with all the metal ions and dinucleotide substrates used. With Cd^2+ as the divalent metal ion, inactivation of the enzyme occurs whether enzyme alone is present or is turning over. Upon inactivation only Cd^2+ ions are bound to the enzyme which becomes denatured. Modification of the enzyme to give an SCN-enzyme decreases the ability of Cd^2+ to cause inactivation. The modified enzyme generally exhibits increases in K_NAD and K_i_metai and decreases in V_max as the metal size increases from Mg^2+ to Mn^2+ or Cd^2+, indicative of crowding in the site. In all cases, affinity for malate greatly decreases, suggesting that malate does not bind optimally to the modified enzyme. For the native enzyme, primary deuterium isotope effects increase with a concomitant decrease in the 13-C effects when NAD is replaced by an alternate dinucleotide substrate different in redox potential. This suggests that when the alternate dinucleotides are used, a switch in the rate limitation of the chemical steps occurs with hydride transfer more rate limiting than decarboxylation. Deuteration of malate decreases the 13-C effect with NAD for the native enzyme, but an increase in 13-C effect is obtained with alternate dinucleotides. These suggest the presence of a secondary 13-C effect in the hydride transfer step. This phenomenon is also applicable to the modified enzyme with NAD as the substrate.

Type 2 diabetes is a major health concern worldwide. One of its complications is postprandial hyperglycemia, i.e., high blood glucose concentrations, caused by glucose fast release from dietary polysaccharides into the bloodstream after meals. α-Glucosidase inhibitor drugs reduce postprandial hyperglycemia by inhibiting maltase/glucoamylase (MGAM) and sucrase/isomaltase (SI). MGAM and SI transform polysaccharides into absorbable monosaccharides, and inhibiting them delays monosaccharide release into the blood. The three commercially available α-glucosidase inhibitors are limited by their absorption abilities, inhibition efficacies, and side effects, which highlights the need for more specific α-glucosidase inhibitors. Because enzymes catalyze their reactions by tightly binding to their cognate transition states (TS), TS analogs can be powerful inhibitors and potential drugs. The measurement and interpretation of kinetic isotope effects (KIEs) is the only method that can directly determine TS structures on large molecules. In this work, methods to prepare radioisotopically labelled maltose were developed, as well as methods to measure KIEs on acid- and enzyme-catalyzed maltose hydrolysis. However, the methods developed did not achieve the required precision for TS analysis. Also, KIEs were calculated computationally for a model reaction of maltose hydrolysis. / Thesis / Master of Science (MSc)

This thesis describes a method for calculating lake evaporation as a proportion of water inputs (E/I) for large surface water bodies, using stable isotope ratios of oxygen (18O/16O) and hydrogen (2H/1H) in water. Evaporation as a proportion of inflow (E/I) is calculated for each Laurentian Great Lake using a new dataset of 516 analyses of δ18O and δ2H in waters sampled from 75 offshore stations during spring and summer of 2007. This work builds on previous approaches by accounting for lake effects on the overlying atmosphere and assuming conservation of both mass and isotopes (18O and 2H) to better constrain evaporation outputs. Results show that E/I ratios are greatest for headwater Lakes Superior and Michigan and lowest for Lakes Erie and Ontario, controlled largely by the magnitude of hydrologic inputs from upstream chain lakes. For Lake Superior, stable isotopes incorporate evaporation over the past century, providing long-term insights to the lake’s hydrology that may be compared to potential changes under a future – expectedly warmer – climate. Uncertainties in isotopically derived E/I are comparable to conventional energy and mass balance uncertainties. Isotope-derived E/I values are lower than conventional energy and mass balance estimates for Lakes Superior and Michigan. The difference between conventional and isotope estimates may be explained by moisture recycling effects. The isotope-based estimates include only evaporated moisture that is also advected from the lake surface, thereby discounting moisture that evaporates and subsequently reprecipitates on the lake surface downwind as recycled precipitation. This shows an advantage of applying an isotope approach in conjunction with conventional evaporation estimates to quantify both moisture recycling and net losses by evaporation. Depth profiles of 18O/16O and 2H/1H in the Great Lakes show a lack of isotopic stratification in summer months despite an established thermocline. These results are indicative of very low over-lake evaporation during warm summer months, with the bulk of evaporation occurring during the fall and winter. This seasonality in evaporation losses is supported by energy balance studies. For Lakes Michigan and Huron, the isotope mass balance approach provides a new perspective into water exchange and evaporation from these lakes. This isotope investigation shows that Lake Michigan and Lake Huron waters are distinct, despite sharing a common lake level. This finding advocates for the separate consideration of Lake Michigan and Lake Huron in future hydrologic studies.