[NiFe] hydrogenases are microbial metalloenzymes that catalyse the reversible interconversion between molecular hydrogen and protons with high selectivity and efficiency. The catalytic properties of different [NiFe] hydrogenases vary according to the physiological roles they each play, yet all seem to be based upon an almost identical catalytic site architecture. Through efforts to understand the structural and mechanistic basis for the differing properties of [NiFe] hydrogenases, it has become increasingly evident that electron transfer to and from the active site, mediated by a set of Iron-Sulphur clusters, influences to a significant extent the observed catalytic properties of different hydrogenases. Here we present a comprehensive study of E. coli Hyd-1, an O₂-tolerant hydrogenase, by PFE with a focus on the properties that are characteristic of O₂-tolerant enzymes: overpotential requirement, lack of H₂ production, low K^H₂<sub style='position: relative; left: -1.2em;'>M</sub>, and high E_switch. We show that Hyd-1 catalysis can be made reversible by increasing the equilibrium potential for the reaction through changes in substrate concentration, and that electron transfer into and out of the enzyme molecule, rather than active site properties, is responsible for the characteristics of overpotential and bias in Hyd-1. We present a set of experiments with Hyd-2 from E. coli in which surface-exposed cysteine residues are specifically introduced near the distal and medial Iron-Sulphur clusters to act as points of attachment for photosensitizer molecules, and a study of the kinetics of electron injection from photoexcited molecules to the enzyme and subsequent absorbance changes attributed to transient redox changes at the active site. We are able to show lightdependent H2 production from a Hyd-2 + photosensitizer system. Finally, we present the first purification of the formate-hydrogen lyase (FHL) complex from E. coli, the complex responsible for H2-production by this organism during fermentation, and we provide a characterisation of the complex by EPR and PFE. The properties of Hyd-3, the hydrogenase subunit of the FHL, seem to differ from those observed previously for other [NiFe] hydrogenases.

Biocatalysis is an ever evolving field that uses enzymes or microorganisms for chemical synthesis. By utilizing enzymes that generally have evolved for specific reactions under mild conditions and temperatures, biocatalysis can be a more environmentally friendly option compared to traditional chemistry. Amide-type chemistries are important and bond formation avoiding poor atom economy is of high priority in organic chemistry. Biocatalysis could potentially be a solution but restricted substrate scope is a limitation. Esterases/lipases usually display broad substrate scope and catalytic promiscuity but are poor at hydrolyzing amides compared to amidases/proteases. The difference between the two enzyme classes is hypothesized to reside in one key hydrogen bond present in amidases, which facilitates the transition state for nitrogen inversion during catalysis. In this thesis the work has been focused on introducing a stabilizing hydrogen bond acceptor in esterases, mimicking that found in amidases, to develop better enzymatic catalysts for amide-based chemistries. By two strategies, side-chain or water interaction, variants were created in three esterases that displayed up to 210-times increased relative amidase specificity compared to the wild type. The best variant displayed reduced activation enthalpy corresponding to a weak hydrogen bond. The results show an estimated lower limit on how much the hydrogen bond can be worth to catalysis. MsAcT catalyze kinetically controlled N-acylations in water. An enzymatic one-pot one-step cascade was developed for the formation of amides from aldehydes in water that gave 97% conversion. In addition, engineered variants of MsAcT with increased substrate scope could synthesize an amide in water with 81% conversion, where the wild type gave no conversion. Moreover, variants of MsAcT displayed up to 32-fold change in specificity towards amide synthesis and a switch in reaction preference favoring amide over ester synthesis. / <p>QC 20161125</p>

The kinetic mechanism of activation of the NAD-malic enzyme by fumarate and the transition state structure for the oxidation malate for the NAD-malic enzyme reaction have been studied. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:Mg:NAD:malate complexes. The activation by fumarate results in a decrease in K_imalate and an increase in V/K_malate by about 2-fold, while the maximum velocity remains constant. A discrimination exists between active and activator sites for the binding of dicarboxylic acids. Activation by fumarate is proposed to have physiologic importance in the parasite. The hydride transfer transition state for the NAD-malic enzyme reaction is concerted with respect to solvent isotope sensitive and hydride transfer steps. Two protons are involved in the solvent isotope sensitive step, one with a normal fractionation factor, another with an inverse fractionation factor. A structure for the transition state for hydride transfer in the NAD-malic enzyme reaction is proposed.

Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the (βγ-bridge position of pyrophosphate to a (β-nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi. Neither back-exchange by [32p] nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction. Reduction of the pyridoxal 5'-phosphate-inactivated enzyme with NaB[3H]4 indicates that about 7 lysines are modified in free enzyme and fructose 1,6-bisphosphate protects 2 of these from modification. The pH dependence of the enzyme-reactant dissociation constants suggests that the phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, inorganic phosphate, and Mg-pyrophosphate must be completely ionized and that lysines are present in the vicinity of the 1- and 6-phosphates of the sugar phosphate and bisphosphates probably directly coordinated to these phosphates. The pH dependence of kinetic parameters suggests that the enzyme catalyzes its reaction via general acid-base catalysis with the use of a proton shuttle. The base is required unprotonated in both reaction directions. In the direction of fructose 6-phosphate phosphorylation the base accepts a proton from the hydroxyl at C-l of F6P and then donates it to protonate the leaving phosphate. The maximum velocity of the reaction is pH independent in both reaction directions while V/K profiles exhibit pKs for binding groups (including enzyme and reactant functional groups) as well as pKs for enzyme catalytic groups. These data suggest that reactants bind only when correctly protonated and only to the correctly protonated form of the enzyme.

As enzimas desempenham papéis essenciais no metabolismo celular. A ausência ou inativação delas podem trazer sérios prejuízos para o desempenho fisiológico de um organismo. Algumas enzimas são comuns a todos os organismos, outras, por sua vez, podem estar ausentes em determinados grupos biológicos, o que pode representar um excelente ponto de partida para estudos bioquímicos que objetivam o desenvolvimento de fármacos mais efetivos. Nesse sentido, é destacado neste trabalho um grupo de enzimas conhecidas como Old Yellow Enzyme (OYE), que são comumente encontradas em bactérias, leveduras, plantas, protozoários, mas estão ausentes em mamíferos. Conhecer a relação estrutura-função dessas proteínas pode trazer alternativas para tratamentos de doenças nas quais o agente etiológico depende da funcionalidade de uma OYE. Dessa forma, doenças negligenciadas, como as leishmanioses e doença de Chagas tornam-se um alvo interessante para o estudo dessas proteínas. Já foi demonstrado o papel que a OYE de Trypanosoma cruzi (TcOYE) desempenha no metabolismo das prostaglandinas, contudo, os dados sobre o comportamento dessas enzimas frente a vários compostos descritos como substratos e a descrição do papel fisiológico dessas proteínas não é completamente compreendido. Além disso, não há informações estruturais e funcionais sobre a OYE de Leishmania braziliensis (LbOYE) na literatura. Assim, um dos principais objetivos desse trabalho de doutorado foi determinar parâmetros estruturais e funcionais das proteínas LbOYE e TcOYE e analisá-los de forma comparativa. Os resultados apresentados sugerem que a LbOYE foi obtida em sua forma enovelada e que a mesma apresenta uma forma sutilmente mais alongada que sua ortóloga TcOYE. Adicionalmente, os resultados também mostram que a proteína LbOYE apresenta menor estabilidade estrutural e maior flexibilidade comparada à TcOYE. Os ensaios de prospecção de ligantes apresentados nesse trabalho mostraram que as enzimas foram obtidas em sua forma funcional e que o composto menadiona foi o que apresentou menor afinidade de interação dentre os compostos avaliados. Além disso, testes de atividade enzimática em baixa pressão de oxigênio com a proteína TcOYE se mostraram efetivos para determinação de moduladores da atividade funcional da proteína. Este trabalho é pioneiro na caracterização estrutural e funcional da LbOYE. / Enzymes play key roles in cellular metabolism. The absence or inactivation of them can cause serious damage to the physiological performance of an organism. Some enzymes are common to all organisms; others, in turn, may be absent in certain biological groups, which may represent an excellent starting point for biochemical studies aiming at the development of more effective drugs. In this sense, it is highlighted in this work a group of enzymes known as Old Yellow Enzyme (OYE), which are commonly found in bacteria, yeasts, plants, protozoa, but are absent in mammals. Knowing the structure-function relationship of these proteins may provide alternatives for treatments of diseases in which the etiological agent depends on the functionality of an OYE. Thus, neglected diseases such as leishmaniasis and Chagas\' disease become an interesting target for the study of these proteins. However, the role that Trypanosoma cruzi OYE (TcOYE) plays in the metabolism of prostaglandins has been demonstrated, however, data on the behavior of these enzymes against various compounds described as substrates and description of the physiological role of these proteins is not fully understood. In addition, there is no structural and functional information on the OEE of Leishmania braziliensis (LbOYE) in the literature. Thus, one of the main objectives of this doctoral project was to determine structural and functional parameters of LbOYE and TcOYE proteins and to analyze them in a comparative way. The presented results suggest that the LbOYE was obtained in its endovelada form and that it presents a subtly more elongated form than its ortholog TcOYE. In addition, the results also show that the LbOYE protein presents lower structural stability and greater flexibility compared to TcOYE. The ligand prospecting trials presented in this study showed that the enzymes were obtained in their functional form and that the menadione compound had the lowest interaction affinity among the evaluated compounds. In addition, low oxygen pressure enzymatic activity tests with the TcOYE protein proved to be effective for the determination of modulators of the functional activity of the protein. This work is a pioneer in the structural and functional characterization of LbOYE.

La nitrate réductase périplasmique de Rhodobacter sphaeroides possède, un cofacteur à Mo (site actif), un centre [4Fe-4S] et deux hèmes de type c formant une chaîne de transfert électronique intramoléculaire. Ce travail est centré sur deux aspects moléculaires de la catalyse de cette enzyme : la réactivité au niveau du site actif de l’enzyme et les processus de transfert d’électrons intramoléculaires. Ces questions sont abordées en s’appuyant parallèlement sur des approches de mutagénèse dirigée, d’activités enzymatiques, de spectroscopie de résonance paramagnétique électronique (RPE) en onde continue et impulsionnelle et sur des mesures de potentiels redox associés aux cofacteurs de l’enzyme. La première partie de ce travail est consacrée à la caractérisation spectroscopique et physico-chimique d’intermédiaires Mo(V) du site actif afin de déterminer leur structure et leur positionnement possible dans le cycle catalytique. Nous avons ainsi étudiée de manière détaillée deux intermédiaires Mo(V) en présence de nitrate dont nous montrons qu’ils présentent des différences structurales au-delà de la première sphère de coordination du Mo. Dans la seconde partie, nous mettons en évidence le rôle d'un acide aminé très conservé (Lys) dans le transfert d'électrons intramoléculaire. Cet acide aminé chargé positivement est situé dans la seconde sphère de coordination du centre [4Fe-4S] et joue un rôle majeur dans la modulation des propriétés rédox du centre [4Fe-4S], ce qui affecte fortement les propriétés catalytiques de l'enzyme. L’ensemble de nos résultats permettent ainsi d’identifier dans l’environnement du Mo des éléments déterminants dans la réactivité de l’enzyme. / The periplasmic nitrate reductase from Rhodobacter sphaeroides contains, in addition to the Mo-cofactor, a [4Fe-4S] center and two c-type hemes defining an intramolecular electron transfer chain. This work focuses on two molecular aspects of the catalysis: the reactivity of the Mo-cofactor, and the intramolecular electron transfer step. These issues are dealt by combining approaches as site-directed mutagenesis, enzymatic activities, continuous-waves (CW) and pulse electron paramagnetic resonance spectroscopy (EPR), UV-Vis spectroscopy and redox titration of metal cofactors of the enzyme. A first part of this work is devoted to the spectroscopic and physicochemical characterization (thermodynamic and kinetic properties) of Mo (V) intermediates of the active site in order to determine their structure and their catalytic relevance. We have undertaken a detailed characterization of two Mo(V) intermediates generated in presence of nitrate, which display some structural differences beyond the first coordination sphere of the Mo(V) ion. In a second part, we highlight the role of a highly conserved amino acid (Lys) in intramolecular electron transfer. This positively-charged amino acid is located in the second coordination sphere of the [4Fe-4S] center and plays a major role in the redox properties tuning of the [4Fe-4S] center thus strongly affecting the catalytic properties of the enzyme. All together, these data provide some structural insights on the enzyme reactivity beyond the first coordination sphere of the Mo-cofactor.

No description available.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 173-184).

Textile dyes are molecules designed to impart a permanent colour to textile fabrics. They pose an environmental problem because they are toxic and they decrease the aesthetic value of rivers and lakes. Current technologies for dye removal cannot remove all classes of dyes and two or more technologies are usually combined to achieve statisfactory decolourization efficiencies. Lignin-degrading enzymes like laccases are potential technologies for dye decolourization and decolourization with immobilized laccase has been intensively investigated. The majority of those studies however have focused on dye disappearance and several reported that significant dye adsorption had occured during the dye removal, making the role of the enzyme unclear. Moreover, textile wastewaters contain auxiliary chemicals that can impact enzymatic dye decolourization and very few studies have evaluated the impact of those substances on laccase. This research evaluated the feasibility of treating dye-contaminated textile wastewaters with an immobilized laccase system. The first sub-objective was to examined the decolourization of Reactive blue 19 (an anthraquinone dye) by Trametes versicolor laccase immobilized on controlled porosity carrier (CPC) silica beads and the second was to analyze the kinetic effects of a non-ionic surfactant Merpol, sodium sulfate, and sodium chloride on laccase decolourization of Reactive blue 19. Decolourization of Reactive blue 19 by immobilized laccase was mainly enzymatic although dye some adsorption occurred. Decolourization led to less toxic by-products from azo and indigoid dyes whereas increased toxicity was observed for anthraquinone dyes. The feasibility of immobilizing laccase on poly(methyl methacrylate) (PMMA) through its sugar residues with a simple procedure was demonstrated and the mass of enzyme immobilized compared well with other commercial acrylic supports. The decolorization of Reactive blue 19 by laccase was inhibited by the non-ionic surfactant, Merpol by substrate depletion. A model describing this inhibition was developed and was validated by a saturated equilibrium binding experiment. While sodium sulfate (ionic strength) had no effect on either ABTS oxidation or dye decolourization, sodium chloride inhibited laccase during dye decolourization and the type and nature of the inhibition depended on the substrate. With ABTS, the inhibition was hyperbolic non-competitive whereas it was parabolic mixed with Reactive blue 19. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2009-06-16 16:58:47.753

Thesis (Ph.D.)--McMaster University, 2002. / Advisor: R.S. Gupta. Includes bibliographical references. Also available via World Wide Web.