Over the past forty years, there has been an increase in obesity, diabetes, and heart disease, collectively known as metabolic syndrome (MetS), in which fructose has been implicated. In addition to MetS, hereditary fructose intolerance (HFI) has no known treatment aside from the difficult removal of fructose from the diet. Ketohexokinase (KHK) is the first enzyme in the fructose metabolic pathway and catalyzes an ATP-dependent reaction that phosphorylates fructose to fructose 1-phosphate. For effective inhibitor development, it is key to understand the KHK-catalytic mechanism. To that end, the research described in this thesis focuses on two goals: 1) understanding how KHK functions in its role as a metabolic enzyme, using structure-function analysis to inform the development of KHK inhibitors, and 2) investigating how these findings can be used to make KHK a prime therapeutic target for alleviating diseases such as HFI and MetS. The X-ray crystal structure of the mouse-liver isozyme, KHK-C (mKHK-C), was determined at a resolution of 1.79 Å. The mKHK-C structure is in complex with the substrate fructose and the product of catalysis, ADP, forming a ground-state complex. The mKHK-C structure has nearly identical secondary structure to its human homolog and has similar steady-state kinetic parameters validating the use of mouse models for exploring the pre-clinical efficacy of KHK-C inhibitors. Furthermore, six structures of human KHK-C in complex with inhibitors and ligands are presented. These structures support the kinetic analyses showing these inhibitors are all competitive with ATP and reveal the shape and polarity of the ATP-binding pocket to achieve inhibition constants (Ki) as low as 50 nM. Lastly, comparison of all KHK structures demonstrate that the β-sheet domain of KHK is capable of 30.3° rotation of the β-sheet domain towards the active site of the opposing dimer subunit. Kinetic experiments using site-directed mutants of human KHK-C and various viscogens confirmed that a conformational change is linked to KHK’s catalytic function. This research provides a foundation for further development of more specific KHK inhibitors aimed at HFI and MetS therapies. / 2022-10-30T00:00:00Z

This thesis presents a detailed description and analysis of the circular dichroism studies performed on the aromatic residues of fructose 1, 6-bisphosphatase (FbPase) from turkey liver under various experimental conditions. Circular dichroism studies performed on the aromatic residues of FbPase indicate that the presence of the substrate, fructose 1, 6-bisphosphate (FbP) and/or the allosteric inhibitor, adenosine monophosphate (AMP) as well as changes in the pH of the medium produce significant effects on the conformation of the enzyme. The effect of the inhibitor, AMP, on the conformation of the enzyme is more pronounced than that of the substrate, FbP.

There has been progress towards elucidating the mechanism of Escherichia coli glycerol kinase (EcGK) control by its allosteric effectors fructose-1,6-bisphosphate (FBP) and IIAglc (a member of the phosphoenolpyruvate:glycose phosphotransferase system). Determining the mechanism requires analysis of the interaction between these effectors and the substrates of EcGK. In this study, a structural and kinetic approach was used to determine inhibition by both the effectors. For this work, the use of fluorescence anisotropy to observe ligand binding was investigated. Also, a foundation was laid for future NMR experiments with EcGK. For fluorescence studies, E36C EcGK was labeled with fluorescein and tested for changes in anisotropy in the presence of different ligands. To ensure that E36C was an appropriate representative of wildtype protein, initial velocity, inhibition, and heterotropic coupling assays were performed. Groundwork for future NMR experiments required analyzing substitutions of the native EcGK cysteines by initial velocity and inhibition studies. By comparing wildtype enzyme and E36C (variant of wildtype with an engineered cysteine residue at position 36), it was found that E36C is a suitable substitute and was not drastically affected by labeling with fluorescein. Anisotropy values differed upon binding of different ligands and enabled titrations of the enzyme substrate complexes with both effectors to obtain dissociation constants. This supports using the stopped-flow method to assess the on- and off- rates of substrates and to obtain values for Q coupling. Furthermore, the results for FBP showed that inhibition by FBP is K-type (affects affinity) with respect to ATP and V-type (affects enzyme velocity) with respect to ADP. The findings presented also showed that native cysteine substitutions effect some of the catalytic and allosteric parameters of EcGK and would be powerful reporters for ligand binding in NMR. However, the enzymes are unstable and new protocols for protein isolation will need to be drafted.

Dissertation (Ph.D.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.

Thesis (Ph. D.)--Ohio State University, 1927. / Autobiography.

Orientador: Cleide Enoir Petean Trindade / Resumo: O metabolismo da frutose, bem como o seu nível sangüíneo em recém-nascidos não está bem esclarecido. A frutose é uma hexose encontrada normalmente no organismo humano e tem seu metabolismo associado à glicose e ao sorbitol. As principais fontes de frutose são os vegetais e o mel. O leite materno não contém frutose. O metabolismo desse açúcar é independente da insulina o que o torna uma boa opção para utilização em pacientes com deficiência desse hormônio. Além da independência da insulina, o metabolismo da frutose é caracterizado por uma rápida fosforilação e rápida conversão em glicogênio e glicose ou conversão em glicerol, para posterior utilização no metabolismo lipídico. A frutose pode ser produzida endogenamente, a partir da glicose, através da via do sorbitol. Apesar de ser utilizado há tempos clinicamente como uma alternativa à glicose, existem poucos trabalhos na literatura determinando os níveis normais em humanos. Isso ocorreu em grande parte devidas dificuldades na dosagem desse carboidrato: em virtude de ser difícil a sua diferenciação da glicose, outra hexose, além da frutose ser encontrada em pouca quantidade em fluídos orgânicos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The metabolism of fructose as well the blood-levels of fructose in newborn infants are not yet well established. Fructose is an hexose found naturally in fruits, vegetables and honey and its metabolism is associated with glucose and sorbitol. The human milk does not contain fructose. The metabolism of fructose is insulin independent, which makes it an alternative to glucose. Besides its independence of insulin, fructose is rapidly metabolized primarily in the liver, where occurs phosphorylation and conversion to glycogen and glucose or to glycerol, to further utilization in lipid metabolism. Fructose, also, can be produced by the human organism, originating from glucose by the sorbitol pathway. Although fructose is utilized since 1893 for medical purposes, there are few studies in the literature about normal levels of fructose in humans. This lack of studies is due in part to difficulties to determine this sugar in human fluids: glucose interferes in the final results and levels of fructose in biological fluids are very low. Our main goal was to establish the normal levels of fructose in newborn infants at 48 hours of life, with adequate weight for gestational age, breast-fed exclusively and to correlate the level of fructose with the levels of glucose and sorbitol. For this purpose we used the High performance liquid chromatography was used. Our study group was selected among breast-fed term newborn... (Complete abstract click electronic access below) / Mestre

Body adiposity is generally considered the most pertinent factor in puberty attainment; however, recent data suggests that pre-pubertal reproductive tract development may be altered by dietary sugar consumption. Two experiments were conducted to delineate the direct effects of fructose on the maturation of the pre-pubertal reproductive tract and fertility. At three weeks of age, forty gilts were placed on one of five dietary treatments (n=8) containing 15% fat (FAT), 35% fructose (FRU), both fat and fructose (HFHF), or two different controls: one standard industry (IND) diet meant to result in optimal lean growth and a second diet to account for the reduced lysine (LYS) intake in the treatment diets. Body weights did not differ amongst any of the five treatments on the day of sacrifice (P=0.32). As a percentage of BW, total reproductive tracts were heavier in fructose fed gilts (1.3±0.1 v. 0.8±0.1%; P=0.01) compared to non-fructose gilts. In the second experiment, starting at 130d of age, gilts were checked twice daily for puberty attainment. Gilts that attained puberty were artificially inseminated (AI) on their third estrous cycle. On gestational day 38±3, pregnant gilts were harvested for reproductive tract collection. Fewer fructose fed (FRU and HFHF) pigs became pregnant than non-fructose fed (IND, LYS, and FAT) gilts (25% v. 75% respectively; P=0.03). All HFHF gilts failed to become pregnant. Placental weights were greater in LYS fetuses than FAT fetuses (79.07 ± 6.55g v. 47.26 ± 6.45g, respectively, P= 0.04). Taken together, these results demonstrate that fructose consumption increases reproductive tract size, but that reproductive capabilities are reduced. / Master of Science

Résumé La prédominance de l'obésité qui touche les enfants et les adultes a augmenté dans le monde entier ces dernières décennies. Les différentes études épidémiologiques ont prouvé que l'obésité est devenue une préoccupation profonde de santé aux États-Unis et au Canada. Il a été montré que l'obésité a beaucoup d’effets sur la santé ainsi il serait important de trouver différentes causes pour le gain de poids. Il est clair que l'obésité soit la condition de multiples facteurs et implique des éléments génétiques et environnementaux. Nous nous concentrons sur les facteurs diététiques et particulièrement le fructose où sa consommation a parallèlement augmenté avec l'augmentation du taux d'obésité. La forme principale du fructose est le sirop de maïs à haute teneur en fructose (HFCS) qui est employé en tant qu'édulcorant primordial dans la plupart des boissons et nourritures en Amérique du Nord. Il a été suggéré que la prise du fructose serait probablement un facteur qui contribue à l’augmentation de la prédominance de l'obésité. L'objectif de cette étude était d'évaluer s'il y a un rapport entre la consommation du fructose et le risque d'obésité. Nous avons travaillé sur deux bases de données des nations Cree et Inuit. Nous avons eu un groupe de 522 adultes Cree, (263 femmes et 259 hommes) dans deux groupes d'âge : les personnes entre 20 et 40 ans, et les personnes de 40 à 60 ans. Nous les avons classés par catégorie en quatre groupes d'indice de masse corporelle (IMC). L'outil de collecte de données était un rappel de 24 heures. En revanche, pour la base de données d'Inuit nous avons eu 550 adultes (301 femmes et 249 hommes) dans deux groupes d'âge semblables à ceux du Cree et avec 3 catégories d’indice de masse corporelle. Les données dans la base d'Inuit ont été recueillies au moyen de deux rappels de 24 heures. Nous avons extrait la quantité de fructose par 100 grammes de nourriture consommés par ces deux populations et nous avons créé des données de composition en nourriture pour les deux. Nous avons pu également déterminer les sources principales du fructose pour ces populations. Aucun rapport entre la consommation du fructose et l’augmentation de l’indice de masse corporelle parmi les adultes de Cree et d'Inuit n’a été détecté. Nous avons considéré l’apport énergétique comme facteur confondant potentiel et après ajustement, nous avons constaté que l'indice de masse corporelle a été associé à l’apport énergétique total et non pas à la consommation du fructose. Puisque dans les études qui ont trouvé une association entre la consommation de fructose et l’obésité, le niveau de la consommation de fructose était supérieure à 50 grammes par jour et comme dans cette étude ce niveau était inférieur à cette limite (entre 20.6 et 45.4 g/jour), nous proposons que des effets negatifs du fructose sur la masse corporelle pourraient être testés dans des populations à plus haute consommation. Les essais cliniques randomisés et éventuelles études cohortes avec différents niveaux de consommation de fructose suivis à long terme pourraient aussi être utiles. Mots clés : fructose, sirop de maïs à haute teneur en fructose (HFCS), obésité et poids excessif / Summary The prevalence of obesity has increased worldwide in recent decades in both children and adults. Different epidemiologic studies have shown that obesity has become a serious health concern in United States and Canada. It has been proved that obesity has many adverse health outcomes so it is important to identify the different causes of weight gain. It is clear that obesity is a multifactor condition and involves both genetic and environmental elements. In this study, we focus on dietary factors, specifically the consumption of fructose that has increased in parallel to the increase in the obesity rate. The main form of fructose in the diet is high fructose corn syrup (HFCS) that is used principally as a sweetener in most beverages and foods in North America. It has been suggested that the intake of fructose may possibly be a contributing factor to the increased incidence of obesity. The objective of this study was to assess if there is a relationship between consumption of fructose and risk of obesity. We worked on two databases. The first database contained 24-hour recall data collected from a sample of 522 Cree adults (263 women and 259 men), divided into two age groups: people between 20 and 40 years old, and people from 40 to 60 years old. We categorized them into four body mass index (BMI) groups. The second database contained data from two 24-hour recalls administered to 550 Inuit adults (301 women and 249 men). These adults were divided into two age groups similar to Cree and with three BMI categories. The amount of fructose per 100 grams of food consumed by these two samples was calculated and we created food composition data for both. We also determined the main sources of fructose in these populations that was sugar sweetened beverages. Based on our results, we could not detect any relationship between consumption of fructose and an increase in BMI among Cree and Inuit adults. We considered energy intake as a potential cofounding factor and, after adjustment, we found that BMI was associated with total energy intake and not with the consumption of fructose. Since in studies that have found this association the level of fructose consumption was more than 50 grams per day but in this study, this level was lower than this limit ( from 20.6 to 45.4 g / day) , we suggest that negative effects of fructose on body weight may appear only at higher dose. Randomized clinical trials and prospective cohort studies using different levels of consumption with long term follow up could be useful. Key words: Fructose, High Fructose Corn Syrup (HFCS), Obesity, and Overweight

by Lee Tin-wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 79-84). / Abstracts in English and Chinese. / Chapter 1. --- Introduction / Chapter 1.1 --- Chemical sensors --- p.1 / Chapter 1.2 --- Quartz crystal microbalance --- p.5 / Chapter 1.3 --- Film immobilization technologies --- p.11 / Chapter 1.4 --- Research Outlines --- p.13 / Chapter 2. --- Saccharide detection by affinity mass sensor / Chapter 2.1 --- Concept of affinity mass sensor --- p.15 / Chapter 2.1.1 --- Affinity chromatography --- p.15 / Chapter 2.1.2 --- Basis of affinity mass sensor --- p.17 / Chapter 2.1.3 --- Saccharide sensing --- p.19 / Chapter 2.2 --- Experimental --- p.20 / Chapter 2.2.1 --- Flow-through cell --- p.21 / Chapter 2.2.2 --- QCA 917 quartz crystal analyzer --- p.21 / Chapter 2.2.3 --- Experimental setup --- p.25 / Chapter 2.2.4 --- Sensor fabrication --- p.29 / Chapter 2.2.5 --- Analysis procedures --- p.29 / Chapter 2.3 --- Results and Discussion --- p.30 / Chapter 2.3.1 --- Formation of boronate complex --- p.30 / Chapter 2.3.2 --- Response curve --- p.31 / Chapter 2.3.3 --- Ligand (APBA) immobilization --- p.32 / Chapter 2.3.4 --- Effect of various operating parameters --- p.35 / Chapter 2.3.5 --- Calibration and reproducibility --- p.38 / Chapter 2.3.6 --- Kinetics analysis --- p.39 / Chapter 2.3.7 --- Stability of sensor --- p.44 / Chapter 2.3.8 --- Determination of fructose in real samples --- p.44 / Chapter 2.3.9 --- Comparison with conventional saccharides sensors --- p.46 / Chapter 2.4 --- Summary --- p.47 / Chapter 3. --- Sol-gel fabrication of affinity mass sensor / Chapter 3.1 --- Principle of sol-gel method --- p.48 / Chapter 3.2 --- Encapsulation of organic molecules in sol-gel matrices --- p.51 / Chapter 3.3 --- Experimental --- p.53 / Chapter 3.3.1 --- Preparation of alkoxide solutions --- p.53 / Chapter 3.3.2 --- Film deposition on QCM --- p.55 / Chapter 3.3.3 --- Film characterization and surface analysis --- p.56 / Chapter 3.4 --- Results and Discussion --- p.57 / Chapter 3.4.1 --- Optimization of conditions for sol-gel process --- p.57 / Chapter 3.4.1.1 --- Choice of catalyst --- p.57 / Chapter 3.4.1.2 --- "H2O: TEOS ratio, R" --- p.59 / Chapter 3.4.1.3 --- Ligand loading --- p.60 / Chapter 3.4.1.4 --- Surface active agent --- p.60 / Chapter 3.4.1.5 --- Temperature --- p.61 / Chapter 3.4.1.6 --- Ageing and drying --- p.62 / Chapter 3.4.2 --- Characterization of APBA encapsulated film --- p.62 / Chapter 3.4.3 --- Performance of the sol-gel derived sensor --- p.65 / Chapter 3.4.3.1 --- Calibration --- p.65 / Chapter 3.4.3.2 --- Stability --- p.66 / Chapter 3.4.3.3 --- Selectivity --- p.68 / Chapter 3.4.4 --- Applicability of the sol-gel derived sensor --- p.69 / Chapter 3.4.5 --- Comparison between sensors fabricated via crosslinking method and the sol-gel method --- p.70 / Chapter 3.4.5.1 --- Surface uniformity --- p.70 / Chapter 3.4.5.2 --- Reproducibility in mass deposition --- p.72 / Chapter 3.4.5.3 --- Stability --- p.72 / Chapter 3.4.5.4 --- Sensitivity towards fructose standard --- p.73 / Chapter 3.4.5.5 --- Comparison of precision and accuracy --- p.73 / Chapter 3.5 --- Summary --- p.75 / Conclusion --- p.77 / References --- p.79 / Titles for tables --- p.85 / Captions for figures --- p.86 / Appendix I --- p.88 / Appendix II --- p.89 / Appendix III --- p.95

Many diseases correlate with abnormal glucose metabolism in cells and organisms. For instance, the human M2 isoform of the glycolytic enzyme pyruvate kinase (M2PYK) plays an important role in metabolic reprogramming of tumour cells whereby aerobic glycolysis or the ‘Warburg effect’ supports cell proliferation by accumulating necessary biomass. By contrast, gluconeogenesis may play an important role, as observed in certain types of trypanosomatid parasites (e.g. the amastigote form of Leishmania major) where anabolism is essential for infectious properties. Hence, these glucose metabolising enzymes are important potential drug targets for cancer and trypanosomiasis. However, many aspects of their regulatory mechanisms are still poorly understood. This thesis describes biochemical and structural studies on M2PYK and on L. major fructose-1,6-bisphosphatase (LmFBPase), providing insights into allosteric mechanisms and structure-based drug design for both enzymes. Human PYKs and LmFBPase were expressed and purified from Escherichia coli, and their kinetics were fully characterised. It was shown that certain amino acids regulate the activity of M2PYK allosterically, but in opposite ways, with some being inhibitors and others activators. X-ray crystallographic structures and biophysical data of M2PYK complexes with alanine, phenylalanine, serine or tryptophan reveal an R-/T-state oscillating model of M2PYK involving a 11° rotation of each subunit. In addition, M2PYK was demonstrated to be a redox-sensitive enzyme. Reducing reagents were shown to help maintain the tetramer and prevent its dissociation, and thereby to activate M2PYK, whereas oxidation and nitrosylation reagents functioned in the opposite sense. Nitrosylation assays showed that the main nitrosylated residue is Cys326 of M2PYK, which is located on the tetramer interface. Dynamics and modulator effects of PYKs were further studied by hydrogen–deuterium exchange by mass spectrometry. These observations highlight the important effects of amino acids on M2PYK regulation. M1PYK by contrast, was demonstrated to be a constitutively fully active pyruvate kinase, with minor effects from modulators. The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is a potential drug target against leishmaniasis. Here we present biochemical and structural studies for LmFBPase, by characterising its activity in a metal-dependent reaction, as well as its inhibition by AMP. The crystal structure of LmFBPase is a homotetramer, composed of monomers with alternating α/β/α/β/α ‘club sandwich’ topologies. In comparison with previously revealed LmFBPase structures, the AMP-complexed structure shows a rotated form of the tetramer. Comparisons of the structures reveal an ‘unlock-androtate’ allosteric mechanism in which AMP binding causes a series of structural changes culminating in an incomplete and non-productive active site. The structure of the effector site of LmFBPase shows a different conformation from human FBPases, thereby offering a potential specific target for Leishmania.