The cytochromes P450 (CYPs) are a superfamily of biological catalysts that are ubiquitous throughout the biological domain. CYPs are heme-b containing monooxygenases which oxidize substrates with the help of accessory redox partners. CYP substrates include endogenous compounds required for many biological functions and homeostasis, such as steroids, as well as the majority of clinically used drugs and environmental xenobiotics. The majority of studies that have been performed to date are on P450cam (CYP101) from Pseudomonas putida. Of the numerous reactions catalyzed by CYPs, unactivated carbon-carbon bond cleavage, is one of particular versatility. Being unique in their catalytic mechanisms, the C-C bond cleaving enzymes and in particular CYP51 from Mycobacterium tuberculosis are though to be capable of utilizing multiple reactive oxygen intermediates. During the process of C-C bond cleavage, CYP51 catalyzes two classical hydroxylation reactions. The final reaction culminates in an enigmatic third step which cleaves a C-C bond, liberates formate, and installs a 14,15 double bond within its steroid substrate. The mechanism of CYP51s final step is still unclear and the exact activated oxygen species has yet to be observed. CYP51 is also distinct from most CYPs owing to the fact that the acid functionality of the conserved active site “acid-alcohol pair” found in most CYPs, is replaced by a histidine. This study aimed to trap and characterize dioxygen reactive intermediates, and elucidate the role of the unique acid-alcohol pair in the formation and stabilization of these intermediates. This study demonstrates our success in generating, stabilizing, and spectroscopically characterizing reactive dioxygen intermediates in Mtb CYP51. As the life-time of the oxyferrous intermediate in Mtb CYP51 is extremely short at ambient temperatures, this work has shown the laboratory’s expertise in being able to generate reduced oxyferrous intermediates at cryogenic temperatures. These intermediates have only been generated in a handful of cytochromes P450 and as such this work adds critical information to the small body of work currently reported.

Le procédé de captage du CO2 en post-combustion par lavage aux amines est actuellement le plus mature pour réduire les émissions de dioxyde de carbone industrielles. Cependant, s'il existe de nombreux démonstrateurs, son coût en termes d'investissement et de fonctionnement est encore trop important pour être mis en oeuvre à une large échelle. La cinétique de réaction amine-dioxyde de carbone est un des principaux facteurs influençant ces coûts. Les objectifs de ces travaux portent sur l'étude et la compréhension de la cinétique de réaction amine-CO2 et de la mise en place d'un modèle structure-propriété prédictif. Cette démarche est adaptée au grand nombre d'amines envisageables pour le procédé. Dans un premier temps nous avons étudié cinq types d'amines (primaires, secondaires acycliques, secondaires cycliques, tertiaires et multi-amines) représentatifs des molécules candidates. Parmi ces molécules deux comportements peuvent être distingués : d'une part les amines qui forment des carbamates et d'autre part celles qui n'en forment pas. Des mesures réalisées sur des solutions diluées d'amine, à différentes concentrations et à 25°C, obtenues par la technique d'écoulement bloqué ont permis de caractériser la cinétique intrinsèque de chacune des 87 amines par deux constantes cinétiques. Pour chaque type d'amine les principaux facteurs structuraux, électroniques et géométriques influant sur la cinétique de réaction ont été identifiés. Un modèle statistique utilisant des descripteurs moléculaires pour décrire les différents paramètres de chaque amine a permis d'établir une relation structure - propriété pour différentes constantes cinétiques. Un nouveau descripteur de l'encombrement stérique de l'azote a également été calculé pour décrire les résultats et prédire la réactivité des amines / The post-combustion process by amine scrubbing is currently the most mature to reduce carbon dioxide emissions from industry. However, if there are numerous demonstrators, the investment and operating cost of this process are still too important to develop it in a large scale. The kinetics of reaction between the amine and the carbon dioxide is one of the major factor which influence the costs. The objectives of this work are to study and understand the kinetics of the amine-CO2 reaction and to set up of a predictive structure-property model. This approach is adapted to the large number of possible amines which can be candidates for the process. In a first time we study five kinds of amines (primary, acyclic secondary, cyclic secondary, tertiary and multi-amines) representatives of candidate molecules. Among those molecules, two behaviours can be distinguished: one the one hand amines which form carbamates and on the other hand those which do not form carbamates. Measurements have been realised at 25 °C in diluted solutions by stopped-flow technique to characterize the intrinsic kinetics of each of the 87 studied amines using two kinetic constants. For each kind of amine, the main structural factors, electronic and geometric, which impact the kinetics of reaction have been identified. Then, from a statistical model using molecular descriptors to describe the different parameters of each amine, a structure-property relationship has been set up with the different kinetic constants. A descriptor of the steric hindrance has been developed

Supramolecular systems comprised of non-covalent interactions are reversible in nature. This intrinsic reversibility of these systems is essential in achieving several functions, making it crucial to understand the dynamics of supramolecular systems. However, studies on the dynamics of supramolecular systems have always lagged behind structural and thermodynamic characterization of innumerable supramolecular systems developed. The first objective of this work was to understand the dynamics leading to a shift in the acidity constant (pKa) for 2-aminoanthracenium cation (AH+) upon binding with cucurbit[7]uril (CB[7]) host molecule. The adiabatic deprotonation of free AH+ in water was found to be inhibited in the complex with CB[7]. Different spectral characteristics for the protonated and deprotonated form of the guest molecule were used to understand the mechanism of this pKa shift associated with the binding to CB[7]. The results suggested that the pKa shift upon binding with CB[7] is a result of the slowing down of the deprotonation step in the complex, whereas the association rate constant did not change very much. The second objective of this work was to understand the role of cations on the binding dynamics of the N-phenyl-2-naphthyl amine (Ph-A-Np) binding to CB[7]. Ph-A-Np has two binding sites, which can lead to 1:1 and 2:1 host-guest complexes. The results indicate a switch in the binding mechanism for Ph-A-Np at low and high concentration regimes of sodium ions. Sodium ion was found to reduce the binding affinity of the naphthyl group to CB[7] whereas the complex formed by the phenyl group with CB[7] bound to one sodium ion was found to be stabilized. The final objective of this work was to study how structural changes to a guest molecule can affect the binding dynamics for the formation of a 2:1 “capsule” like complex with octa acid (OA). The dissociation for the OA capsule with pyrene (Py) as the encapsulated guest was shown to happen in 2.7 s previously. Two pyrene derivatives, 1-methylpyrene (MePy) and 1-pyrenemethanol (PyMeOH) were chosen as guest molecules to study the effect of these substituents on pyrene on the capsule dissociation dynamics. The results show that the residence time for the guests in the OA capsule depends on the substituents. For PyMeOH and MePy a shorter and longer residence time respectively in the capsule was observed when compared to Py. / Graduate / 2019-09-30

This thesis is focussed on structural and functional studies of a novel glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. GpdQ is highly promiscuous and is the first known phosphatase which is capable of degrading all three classes of phosphate esters (mono-, di- and triesters). Remarkably, GpdQ is also able to hydrolyse stable aliphatic phosphate esters and has been shown to degrade the hydrolysis product of the nerve agent VX. For these reasons, GpdQ has been realised to have potential as a powerful bioremediator for the removal of organophosphate pesticides and nerve agents. GpdQ is a binuclear metallohydrolase in which one of the metal ions is very weakly bound. Chapter 1 introduces the catalytic mechanisms of binuclear metallohydrolases by examining two related phosphate ester-degrading enzymes. Since one of the main features of catalysis addressed in this thesis are the differential metal binding affinities of GpdQ, Chapter 1 also canvasses a range of other binuclear metallohydrolases with similar behaviour. Chapter 2 examines the structural and evolutionary relationship between GpdQ and a number of other related enzymes. Using genome database searches, the two most closely related enzymes are identified. In performing these searches, a novel, putative binuclear metallohydrolase from Homo sapiens is also discovered. This enzyme, Hsa_aTRACP, is most closely related to PAPs, however construction of a homology model indicates that the active site tyrosine residue of PAP is replaced by histidine. In this respect, it may represent an evolutionary link to Ser/Thr protein phosphatases and GpdQ. The biology and chemistry of this putative enzyme is discussed. PAPs are the only binuclear enzymes with an established heterovalent active site of the type Fe(III)-M(II) (where M=Fe, Zn or Mn) whereas the majority of enzymes in this family have homovalent metal centres, including GpdQ and Ser/Thr protein. This is brought about due to the nature of the coordination sphere imposed by the enzyme. The activity of GpdQ can be reconstituted in the presence of Co(II), Zn(II), Mn(II) and Cd(II). Chapter 3 examines the kinetic properties of a binuclear homovalent system by studying the kinetic properties of Cd(II)-substituted GpdQ and a corresponding model complex. This comparative study leads to the identification of a terminal hydroxide molecule as the likely reaction-initiating nucleophile in Cd(II)-GpdQ with a pKa of 9.4. In Chapter 4, a detailed study of the structural, kinetic and spectroscopic behaviour of Co(II)-substituted GpdQ is presented. This chapter specifically probes the formation of the binuclear active site, the role of the metal ions in catalysis, the identity of the nucleophile and the potential role of any first or second coordination sphere residues in the regulation of enzyme activity, proton donation and metal ion coordination. Based on these findings, a detailed reaction mechanism is proposed in which the substrate itself promotes the formation of the catalytically competent binuclear centre and phosphorolysis occurs following nucleophilic attack by a terminal hydroxide molecule. A potential role of Asn80 (a ligand of one of the metal ions) in regulating both substrate and metal binding, and the role of the bridging hydroxide molecule in the activation of the terminal nucleophile is proposed. Chapter 5 employs a combination of kinetic and spectroscopic techniques to probe the proposed catalytic mechanism of GpdQ in depth. The formation of the catalytically competent binuclear centre is observed in pre-steady state studies, an integral first step in the catalytic mechanism. The dissociation and rate constants associated with formation of the binuclear centre are quantified. The rate of substrate turnover in GpdQ is relatively modest but is enhanced by a structural rearrangement involving the flexible Asn80 ligand. This structural change fine-tunes the reaction mechanism, leading to optimal reactivity. The steady-state kinetic properties of a series of metal ion derivatives (Co(II), Cd(II) and Mn(II)) of GpdQ and their reactivity towards a number of substrates are also compared. These findings lead to the conclusion that the reaction mechanism of GpdQ is modulated by both substrate and metal ion. In this respect, GpdQ is adaptive to the environmental conditions to which it is exposed by employing a flexible mechanistic strategy to achieve catalysis. Chapter 6 correlates the electronic and geometric structure of the binuclear centre in GpdQ as a means to probe specific aspects of the mechanism. This study uses the wild type enzyme and a site-directed mutant (Asn80Asp) to examine the structure of the metal ions at two stages of catalysis. The role of the bridging hydroxide molecule in nucleophilic activation is specifically addressed by monitoring changes in the electronic exchange interaction and other structural parameters as a result of phosphate binding. Also, the coordination environment of the metal ions in both the free enzyme and the phosphate-bound enzyme of wild type and Asn80Asp GpdQ were assessed against the currently proposed structures. The findings in this chapter corroborate the proposed catalytic mechanism of GpdQ. In summary, this project led to a detailed understanding of the mechanism of GpdQ, and provided insight into how both the metal ion composition and the identity of the substrate may modulate this mechanism. The knowledge gained may lead to the design of catalytically more efficient derivatives (mutants) of GpdQ for application in bioremediation.

The overproduction of reactive oxygen species (ROS) have been linked to diseases and other pathologies. As therapeutic agents, antioxidants have been tested and some shown to attenuate these diseases by relieving oxidative stress. The May laboratory has previously developed a family of phenylaminoalkyl selenides and has demonstrated the antihypertensive and antioxidant properties of these compounds. To further understand the antioxidant property of these selenide compounds, the two step mechanism of the reaction between the selenoxide form and glutathione was investigated by stopped-flow and mass spectrometry, leading to the detection and characterization of a novel thioselenurane intermediate. Mass spectrometry studies supported the redox cycle of the selenide compounds as a straightforward cycle with no byproducts or side reactions and was the first evidence reported of a thioselenurane intermediate present in a reduction reaction of a selenoxide. The therapeutic potential of these compounds was further supported by cell and histological studies demonstrating their ability to alleviate the cardiotoxic effect of anthracyclines without affecting the anti-cancer property of the drugs. Codosage of a phenylaminoethyl selenide with Doxorubicin decreased the infiltration of inflammation cells in the myocardium of mice. Phenylaminoethyl selenides were also able to maintain the body weight of mice treated with Doxorubicin, compared to mice treated with Doxorubicin alone. In order to make the possibility of using Phenylaminoalkyl selenides as therapeutic agents or supplements with other agents, delivery of the compounds was investigated. N acetyl phenylaminoethyl selenides were successfully encapsulated into poly(lactic-co-glycolic) (PLGA) nanoparticles using the nanoprecipitation technique. An attempt was made to demonstrate the ability of these selenide- nanoparticles to reduce cellular oxidative stress caused by incubation with LPS. Future studies are needed to optimize the loading of the selenide compounds into nanocarriers and to demonstrate the ability of the encapsulated drug to work as the free drug. The long term goal of this research is to fully understand the potential of phenylaminoalkyl selenides as an efficient therapeutic agent for ailments derived from increased levels of ROS and a state of oxidative stress. As a supplemental project funded by the National Science Foundation, the Center for Innovative Biomaterial Education and Research (CIBER) was created. Enzymatically catalyzed reaction and polymerizations were investigated using Candida antarctica Lipase B (CALB). Several CALB catalyzed Michael addition reactions were successful and yielded compounds that could be used as future reactants and monomers. As an education requirement of the project a website was created in order to educate the public of the importance, sources and uses of biomaterials. The website provides information for all levels of students and educators. This center has allowed The Georgia Institute of Technology to form relationships and exchange programs with leading universities around the world allowing the exchange of knowledge and research in biomaterials.

Supramolecular systems, which are formed by the noncovalent intermolecular interactions between molecules, are highly dynamic. The high reversibility of supramolecular systems leads to some functional features that cannot be achieved by the single chemical component. The kinetic information for the supramolecular systems can not be inferred from thermodynamic studies or structural studies. Furthermore, the information provided by the dynamic study can be employed to infer or explain the results from the thermodynamic study and the structural study. The first objective of this work was to study the dynamics and the binding mechanism of cucurbit[7]uril with a charged guest molecule (2-naphthyl-1-ethylammonium cation, NpAmH+). In general, the binding affinity of cucurbit[7]uril to the positively charged guests are very high compared with other host systems such as cyclodextrins and bile salt aggregates. In this work, the complexation of cucurbit[7]uril and NpAmH+ was studied from a kinetic point of view. Results showed that the high binding affinity of cucurbit[7]uril to NpAmH+ was due to the high association rate constant and the low dissociation rate constant for the complexation of cucurbit[7]uril and NpAmH+. Moreover, the competition between co-cations and NpAmH+ for the binding sites of cucurbituril molecules retarded the complexation process for cucurbit[7]uril binding to NpAmH+ and decreased the overall equilibrium constant for the formation of cucurbit[7]uril-NpAmH+ complex. The second objective of this work was to study the chiral recognition observed for the formation of 2:2 complexes between β-cyclodextrin and 2-naphthyl-1-ethanol (NpOH). The binding of β-cyclodextrin and NpOH leads to the formation of two 1:1 complexes and three 2:2 complexes. The binding dynamics of NpOH with β-cyclodextrin in the 1:1 complex is fast and occurs within microseconds. A much slower dynamics was observed for the formation of the 2:2 complex. Results showed that more 2:2 complex were formed for (R)-NpOH than for (S)-NpOH, which is due to the difference of the dissociation rate constant of the 2:2 complex for both NpOH enantiomers. The dissociation rate constant of the 2:2 complex for (R)-NpOH is 46.8% lower than that for (S)-NpOH while the association rate constant of the 2:2 complex are similar for both NpOH enantiomers. The third objective of this work was to study the dynamics and the binding mechanism of octa acid with pyrene. As known from the work of other researchers, the accessibility of small molecules (e.g. I- or O2) to pyrene bound to octa acid is largely limited by the octa acid capsule. In this study, a two-step successive process was observed for the complexation of octa acid and pyrene. The first step, which was related to the formation of octa acid-pyrene 1:1 complex, was sufficiently fast to be viewed as a pre-equilibrium process. The second step, which was related to the formation of octa acid-pyrene 2:1 complex, was slow on the millisecond – second time scale. The high binding affinity of octa acid to pyrene was observed, which is due to the low dissociation rate constant for the octa acid-pyrene 2:1 complex. / Graduate

This thesis is focussed on structural and functional studies of a novel glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. GpdQ is highly promiscuous and is the first known phosphatase which is capable of degrading all three classes of phosphate esters (mono-, di- and triesters). Remarkably, GpdQ is also able to hydrolyse stable aliphatic phosphate esters and has been shown to degrade the hydrolysis product of the nerve agent VX. For these reasons, GpdQ has been realised to have potential as a powerful bioremediator for the removal of organophosphate pesticides and nerve agents. GpdQ is a binuclear metallohydrolase in which one of the metal ions is very weakly bound. Chapter 1 introduces the catalytic mechanisms of binuclear metallohydrolases by examining two related phosphate ester-degrading enzymes. Since one of the main features of catalysis addressed in this thesis are the differential metal binding affinities of GpdQ, Chapter 1 also canvasses a range of other binuclear metallohydrolases with similar behaviour. Chapter 2 examines the structural and evolutionary relationship between GpdQ and a number of other related enzymes. Using genome database searches, the two most closely related enzymes are identified. In performing these searches, a novel, putative binuclear metallohydrolase from Homo sapiens is also discovered. This enzyme, Hsa_aTRACP, is most closely related to PAPs, however construction of a homology model indicates that the active site tyrosine residue of PAP is replaced by histidine. In this respect, it may represent an evolutionary link to Ser/Thr protein phosphatases and GpdQ. The biology and chemistry of this putative enzyme is discussed. PAPs are the only binuclear enzymes with an established heterovalent active site of the type Fe(III)-M(II) (where M=Fe, Zn or Mn) whereas the majority of enzymes in this family have homovalent metal centres, including GpdQ and Ser/Thr protein. This is brought about due to the nature of the coordination sphere imposed by the enzyme. The activity of GpdQ can be reconstituted in the presence of Co(II), Zn(II), Mn(II) and Cd(II). Chapter 3 examines the kinetic properties of a binuclear homovalent system by studying the kinetic properties of Cd(II)-substituted GpdQ and a corresponding model complex. This comparative study leads to the identification of a terminal hydroxide molecule as the likely reaction-initiating nucleophile in Cd(II)-GpdQ with a pKa of 9.4. In Chapter 4, a detailed study of the structural, kinetic and spectroscopic behaviour of Co(II)-substituted GpdQ is presented. This chapter specifically probes the formation of the binuclear active site, the role of the metal ions in catalysis, the identity of the nucleophile and the potential role of any first or second coordination sphere residues in the regulation of enzyme activity, proton donation and metal ion coordination. Based on these findings, a detailed reaction mechanism is proposed in which the substrate itself promotes the formation of the catalytically competent binuclear centre and phosphorolysis occurs following nucleophilic attack by a terminal hydroxide molecule. A potential role of Asn80 (a ligand of one of the metal ions) in regulating both substrate and metal binding, and the role of the bridging hydroxide molecule in the activation of the terminal nucleophile is proposed. Chapter 5 employs a combination of kinetic and spectroscopic techniques to probe the proposed catalytic mechanism of GpdQ in depth. The formation of the catalytically competent binuclear centre is observed in pre-steady state studies, an integral first step in the catalytic mechanism. The dissociation and rate constants associated with formation of the binuclear centre are quantified. The rate of substrate turnover in GpdQ is relatively modest but is enhanced by a structural rearrangement involving the flexible Asn80 ligand. This structural change fine-tunes the reaction mechanism, leading to optimal reactivity. The steady-state kinetic properties of a series of metal ion derivatives (Co(II), Cd(II) and Mn(II)) of GpdQ and their reactivity towards a number of substrates are also compared. These findings lead to the conclusion that the reaction mechanism of GpdQ is modulated by both substrate and metal ion. In this respect, GpdQ is adaptive to the environmental conditions to which it is exposed by employing a flexible mechanistic strategy to achieve catalysis. Chapter 6 correlates the electronic and geometric structure of the binuclear centre in GpdQ as a means to probe specific aspects of the mechanism. This study uses the wild type enzyme and a site-directed mutant (Asn80Asp) to examine the structure of the metal ions at two stages of catalysis. The role of the bridging hydroxide molecule in nucleophilic activation is specifically addressed by monitoring changes in the electronic exchange interaction and other structural parameters as a result of phosphate binding. Also, the coordination environment of the metal ions in both the free enzyme and the phosphate-bound enzyme of wild type and Asn80Asp GpdQ were assessed against the currently proposed structures. The findings in this chapter corroborate the proposed catalytic mechanism of GpdQ. In summary, this project led to a detailed understanding of the mechanism of GpdQ, and provided insight into how both the metal ion composition and the identity of the substrate may modulate this mechanism. The knowledge gained may lead to the design of catalytically more efficient derivatives (mutants) of GpdQ for application in bioremediation.

This thesis is focussed on structural and functional studies of a novel glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. GpdQ is highly promiscuous and is the first known phosphatase which is capable of degrading all three classes of phosphate esters (mono-, di- and triesters). Remarkably, GpdQ is also able to hydrolyse stable aliphatic phosphate esters and has been shown to degrade the hydrolysis product of the nerve agent VX. For these reasons, GpdQ has been realised to have potential as a powerful bioremediator for the removal of organophosphate pesticides and nerve agents. GpdQ is a binuclear metallohydrolase in which one of the metal ions is very weakly bound. Chapter 1 introduces the catalytic mechanisms of binuclear metallohydrolases by examining two related phosphate ester-degrading enzymes. Since one of the main features of catalysis addressed in this thesis are the differential metal binding affinities of GpdQ, Chapter 1 also canvasses a range of other binuclear metallohydrolases with similar behaviour. Chapter 2 examines the structural and evolutionary relationship between GpdQ and a number of other related enzymes. Using genome database searches, the two most closely related enzymes are identified. In performing these searches, a novel, putative binuclear metallohydrolase from Homo sapiens is also discovered. This enzyme, Hsa_aTRACP, is most closely related to PAPs, however construction of a homology model indicates that the active site tyrosine residue of PAP is replaced by histidine. In this respect, it may represent an evolutionary link to Ser/Thr protein phosphatases and GpdQ. The biology and chemistry of this putative enzyme is discussed. PAPs are the only binuclear enzymes with an established heterovalent active site of the type Fe(III)-M(II) (where M=Fe, Zn or Mn) whereas the majority of enzymes in this family have homovalent metal centres, including GpdQ and Ser/Thr protein. This is brought about due to the nature of the coordination sphere imposed by the enzyme. The activity of GpdQ can be reconstituted in the presence of Co(II), Zn(II), Mn(II) and Cd(II). Chapter 3 examines the kinetic properties of a binuclear homovalent system by studying the kinetic properties of Cd(II)-substituted GpdQ and a corresponding model complex. This comparative study leads to the identification of a terminal hydroxide molecule as the likely reaction-initiating nucleophile in Cd(II)-GpdQ with a pKa of 9.4. In Chapter 4, a detailed study of the structural, kinetic and spectroscopic behaviour of Co(II)-substituted GpdQ is presented. This chapter specifically probes the formation of the binuclear active site, the role of the metal ions in catalysis, the identity of the nucleophile and the potential role of any first or second coordination sphere residues in the regulation of enzyme activity, proton donation and metal ion coordination. Based on these findings, a detailed reaction mechanism is proposed in which the substrate itself promotes the formation of the catalytically competent binuclear centre and phosphorolysis occurs following nucleophilic attack by a terminal hydroxide molecule. A potential role of Asn80 (a ligand of one of the metal ions) in regulating both substrate and metal binding, and the role of the bridging hydroxide molecule in the activation of the terminal nucleophile is proposed. Chapter 5 employs a combination of kinetic and spectroscopic techniques to probe the proposed catalytic mechanism of GpdQ in depth. The formation of the catalytically competent binuclear centre is observed in pre-steady state studies, an integral first step in the catalytic mechanism. The dissociation and rate constants associated with formation of the binuclear centre are quantified. The rate of substrate turnover in GpdQ is relatively modest but is enhanced by a structural rearrangement involving the flexible Asn80 ligand. This structural change fine-tunes the reaction mechanism, leading to optimal reactivity. The steady-state kinetic properties of a series of metal ion derivatives (Co(II), Cd(II) and Mn(II)) of GpdQ and their reactivity towards a number of substrates are also compared. These findings lead to the conclusion that the reaction mechanism of GpdQ is modulated by both substrate and metal ion. In this respect, GpdQ is adaptive to the environmental conditions to which it is exposed by employing a flexible mechanistic strategy to achieve catalysis. Chapter 6 correlates the electronic and geometric structure of the binuclear centre in GpdQ as a means to probe specific aspects of the mechanism. This study uses the wild type enzyme and a site-directed mutant (Asn80Asp) to examine the structure of the metal ions at two stages of catalysis. The role of the bridging hydroxide molecule in nucleophilic activation is specifically addressed by monitoring changes in the electronic exchange interaction and other structural parameters as a result of phosphate binding. Also, the coordination environment of the metal ions in both the free enzyme and the phosphate-bound enzyme of wild type and Asn80Asp GpdQ were assessed against the currently proposed structures. The findings in this chapter corroborate the proposed catalytic mechanism of GpdQ. In summary, this project led to a detailed understanding of the mechanism of GpdQ, and provided insight into how both the metal ion composition and the identity of the substrate may modulate this mechanism. The knowledge gained may lead to the design of catalytically more efficient derivatives (mutants) of GpdQ for application in bioremediation.

The canonical Wnt pathway is a vitally important signalling pathway that plays an important role in cell proliferation, differentiation and fate decisions in embryonic development and in the maintenance of adult tissues. The twelve Armadillo (ARM) repeat-containing protein beta-catenin acts as the signal transducer in this pathway and is continuously degraded in the cytosol by the beta-catenin destruction complex (BDC). Upon receiving the Wnt signal the BDC is inactivated, allowing beta-catenin to accumulate in the cytosol and be transported to the nucleus where it binds to the TCF/LEF family of transcription factors, inducing the expression of cell cycle promotor genes. In this Thesis I describe investigations into the roles of leucine-rich repeat kinase 2 (LRRK2) and the transcription factor TCF7L2 within this signalling pathway. LRRK2 is a large multi-domain protein with strong links to Parkinson’s disease and suggested to play a role in inactivating the BDC in response to the Wnt signal. A recent paper proposed that the previously uncharacterised regions of LRRK2 contain a series of tandem repeat sub-domains. I began an investigation into these sub-domains but I was unable to produce soluble protein constructs despite the use of a range of common techniques, and so I was forced to conclude this project early. The main body of this thesis focuses on the interaction between the intrinsically disordered TCF7L2 and the repeat protein beta-catenin, a very long interface of approximately 4800 Å2 that spans from the third to the eleventh ARM repeat of beta-catenin and residues 12 to 50 of TCF7L2, as determined by X-ray crystal structures. First, a fluorescence reporter system for the binding interaction was developed and used to determine the kinetic rate constants for the association and dissociation of the wild-type construct using stopped-flow fluorescence spectroscopy and time-dependent fluorescence spectroscopy. It was found that association of TCF7L2 and beta-catenin was rapid (7.3 ± 0.1 x107 M-1s-1) with only a single phase was observed, whereas dissociation was biphasic and slow (5.7 ± 0.4 x10-4 s-1, 15.2 ± 2.8 x10-4 s-1). Using either of these two dissociation rate constants the calculated Kd value obtained is much lower than the values previously reported in the literature (8 ± 1 / 20 ± 2 pM compared with 16 nM). This reporter system was then used to investigate the striking variability between three crystal structures previously obtained for the TCF7L2-beta-catenin complex, in which different regions of TCF7L2 show different elements of secondary structure. Mutational analysis revealed that the interface residues on TCF7L2 identified in these structures make little or no contribution to the overall binding affinity, pointing to a transient nature of these contact in solution and suggesting that the observed differences between the structures are due to differences in crystal packing. Further experiments into the effect of osmolarity on the binding equilibrium and kinetics supported this conclusion and suggest a change in the association/dissociation mechanism as a function of ionic strength. Lastly, further mutational analysis of TCF7L2 revealed two regions that contribute particularly strongly to the binding kinetics, suggesting that TCF7L2-beta-catenin assembly proceeds via a two-site avidity mechanism. Some of the most destabilising variants display two additional dissociation phases, indicating the presence of an alternative dissociation pathway that is inaccessible to the wild-type. In summary, the results presented here provide insights into the kinetics of molecular recognition of a long intrinsically disordered region with an extended repeat protein surface, a process shown to involve multiple routes with multiple steps in each.

Stopped-flow experiments in which the NAD-malic enzyme was preincubated with different reactants at near saturating substrate concentrations suggest a slow isomerization of the E:NAD:Mg complex. The lag is eliminated by preincubation with Mg˙² and malate suggesting that the formation of E:Mg:Malate either bypasses or speeds up the slow isomerization step. Circular dichroic spectral studies of the secondary structural changes of the native enzyme in the presence and absence of substrates supports the existence of conformational changes with NAD˙ and malate. Thus, a slow conformational change of the E:NAD:Mg complex is likely one of the rate-limiting steps in the pre-steady state.