Dissertations / Theses on the topic 'Enzyme-Substrate Interactions'

Reaction rates and selectivities were measured for transacylation of fatty acid esters in solvents catalysed by Candida antarctica lipase B and by cutinase from Humicola insolens. With these enzymes classical water-based enzymology can be expanded to many different solvents allowing large variations in interaction energies between the enzymes, the substrates and the surrounding. Further ,hydrolysis reactions catalysed by Bacillus subtilis esterase 2 were investigated. Thermodynamics analyses revealed that the enzyme contribution to reaction rate acceleration compared to acid catalysis was purely entropic. On the other hand, studies of differences in activation entropy and enthalpy between enantiomers and between homologous esters showed that high substrate specificity was favoured by enthalpic stabilisation. Solvent was found to have a profound effect on enzyme catalysis, affecting both reaction rate and selectivity. Differences in substrate solubility will impact enzyme specificity since substrate binding is an equilibrium between enzyme-bound substrate and substrate in free solution. In addition, solven tmolecules were found to act as enzyme inhibitors, showing both competitive and non-competitive behaviour. In several homologous data series enthalpy-entropy compensation relationships were encountered. A possible extrathermodynamic relationship between enthalpy and entropy can easily be lost under co-varying errors propagated from the experiments. From the data in this thesis, one instance was found of a real enthalpy-entropy compensation that could be distinguished from statistical errors, while other examples could not be verified.
QC 20100722

Relatively high enzyme loadings are required for the bioconversion of lignocellulosic biomass, impeding the economical production of cellulosic sugars. The relative stability and robustness of these enzymes make enzyme recycling an attractive cost-reduction strategy. However, the efficiency of enzyme recycling has been limited by the complexity of enzyme-substrate interactions, which are influenced by enzyme, substrate, and physical factors. A lack of techniques to probe specific enzyme adsorption further limits our understanding of these interactions. Therefore, overcoming these challenges to better understand enzyme-substrate interactions is crucial if we are to improve the effectiveness of enzyme recycling strategies. Initial work compared various ways to assess enzyme adsorption during hydrolysis of steam pretreated corn stover (SPCS) using a complete commercial cellulase mixture. While the distribution of six individual enzymes could be followed, the initial approach used was laborious, highlighting the limitations of techniques used to quantify individual enzyme adsorption profiles. A quicker, more sensitive double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was subsequently developed, to follow Cel7A, Cel6A, and Cel7B adsorption during hydrolysis, and shown to agree with earlier results. As enzyme, substrate, and physical factors were known to affect enzyme recycling performance, their influence on individual enzyme adsorption was evaluated. Although the lignin present in the SPCS did not appear to influence enzyme adsorption (although Cel6A adsorbed more readily to the lignin-containing SPCS), cellulose allomorphs and crystallinity did appear to influence enzyme adsorption. The addition of Auxiliary Activity (AA) family 9, an oxidative enzyme, increased desorption of Cel7A, likely by increasing the substrate’s negative charge. The AA9 itself remained primarily in the supernatant, which highlighted the importance of recovering enzymes from both the liquid and solid phases of the reaction. The influence of glucose and ethanol on enzyme adsorption was evaluated, and a reduction in enzyme adsorption was observed at high glucose but not ethanol concentrations. When the addition of fresh substrate was assessed as one way to recover enzymes, by combining enzyme recycling at low glucose concentrations with enzyme supplementation, good overall cellulose hydrolysis (~70%) over 5 rounds of enzyme recycle could be achieved with a 50% reduction in enzyme loading.

Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of protein arginine residues, resulting in the formation of monomethylarginine, and/or asymmetric or symmetric dimethylarginines. Although understanding of the PRMTs has grown rapidly over the last few years, several challenges still remain in the PRMT field. Here, we describe the development of two techniques that will be very useful in investigating PRMT regulation, small molecule inhibition, oligomerization, protein-protein interaction, and substrate specificity, which will ultimately lead to the advancement of the PRMT field. Studies have shown that having an N-terminal tag can influence enzyme activity and substrate specificity. The first protocol tackles this problem by developing a way to obtain active untagged recombinant PRMT proteins. The second protocol describes a fast and efficient method for quantitative measurement of AdoMet-dependent methyltranseferase activity with protein substrates. In addition to being very sensitive, this method decreases the processing time for the analysis of PRMT activity to a few minutes compared to weeks by traditional methods, and generates 3000-fold less radioactive waste. We then used these methods to investigate the effect of truncating the NT of human PRMT1 variant 1 (hPRMT1-V1) on enzyme activity, protein-protein interactions, and substrate specificity. Our studies show that the NT of hPRMT1-V1 influences enzymatic activity and protein-protein interactions. In particular, methylation of a variety of protein substrates was more efficient when the first 10 amino acids of hPRMT1v1 were removed, suggesting an autoinhibitory role for this small section of the N-terminus. Likewise, as portions of the NT were removed, the altered hPRMT1v1 constructs were able to interact with more proteins. Overall, my studies suggest the the sequence and length of the NT of hPRMT1v1 is capable of enforcing specific protein interactions.

Ce travail est consacre a l'etude des mecanismes responsables de la reconnaissance entre la seryl-arnt synthetase d'escherichia coli et son substrat macromoleculaire, l'arnt#s#e#r. La premiere etape de ces travaux a consiste a surexprimer l'arnt#s#e#r#2 et l'arnt#s#e#r ambre suppresseur d'e. Coli. Pour cela, les genes synthetiques des deux arnt#s#e#r ont ete construits par assemblage de sept oligonucleotides chevauchants. L'utilisation d'un plasmide dont le nombre de copies est regule par la temperature permet d'obtenir des cellules bacteriennes contenant un taux d'arnt#s#e#r vingt fois superieur a celui present dans des cellules depourvues de plasmide. Grace a cette surexpression, un protocole de purification comportant deux etapes chromatographiques a pu etre developpe. Les arnt purifies ont ensuite ete utilises pour determiner la contribution du domaine n-terminal de la seryl-arnt synthetase d'e. Coli, a l'efficacite et a la specificite de la reaction d'aminoacylation. Les resultats obtenus, montrent que le domaine n-terminal depourvu de role catalytique lors de la reaction d'activation de l'acide amine, est indispensable, d'une part, au maintien d'une efficacite de catalyse elevee lors du transfert de l'acide amine active sur l'arnt et, d'autre part, a la specificite de reconnaissance des arnt#s#e#r. Parallelement, la mesure des constantes de dissociation de l'arnt#s#e#r#2, revele que la fixation de deux molecules d'arnt sur la seryl-arnt synthetase est regit par un mecanisme de type cooperatif. La derniere partie de ce travail montre que la seryl-arnt synthetase de saccharomyces cerevisiae ne peut etre surexprimee chez e. Coli. Le contexte bacterien ne permettant pas a la seryl-arnt synthetase de s. Cerevisiae d'acquerir une structure tridimensionnelle homogene. Les etudes menees, en parallele sur la seryl-arnt synthetase de s. Cerevisiae surexprimee dans son contexte naturel, revelent une repartition de cette enzyme en deux populations distinctes, et une mobilite electrophoretique, sur sds-page, plus elevee que celle obtenue pour l'enzyme surexprimee chez e. Coli

L’accomplissement de tout acte protéolytique implique nécessairement la formation d'un complexe entre l'enzyme et son substrat. Par différentes approches cinétiques, spectroscopiques et moléculaires nous avons cherché à caractériser les phénomènes mis en jeu au cours de l'hydrolyse, par la thermolysine, de petits peptides en milieu biomimétiques. Cette étude a été conduite à l'interface entre la biochimie, la biophysique, la chimie et la physique. Dans un premier temps, nous nous sommes intéressés au comportement catalytique de la thermolysine sur des substrats modèles et en milieu modifié. Nous avons montré d'une part, que l'ajout d'additifs polyhydroxylés influence grandement l'activité de la thermolysine et d'autre part, affine les connaissances sur la spécificité et la sélectivité de cette enzyme (en particulier, mise en évidence de l'influence du résidu P'2 dans le mécanisme). Dans un deuxième temps, nous présentons des études structurales des peptides substrats en milieu modifié. Nous avons mis en évidence l'absence d'influence du micro-environnement contenant une forte proportion de glycérol sur la conformation des molécules de substrat et le rôle possible de leur structure tridimensionnelle quant à leur hydrolyse. Ces études ont été étendues à un autre modèle peptidique, de forme cyclique ou linéaire, et corrélées aux résultats cinétiques. Dans un troisième temps, par deux approches différentes, nous avons abordé l'étude des relations structure-fonction de la thermolysine. Expérimentalement, par des études cinétiques avec l'enzyme immobilisée et des déterminations de sa structure par spectroscopie laser Raman, nous montrons que l'enzyme est très peu sensible au micro-environnement. Théoriquement en analysant, par modélisation, l'interaction de la thermolysine avec un tripeptide substrat, nous avons mis en évidence des changements de conformation du substrat et/ou des mouvements du site actif enzymatique au cours de l'acte catalytique.

Computational tools play an important role in the description of biological systems. Scientists describe and study structure, conformational changes and interactions between molecules in silico, often as a cheaper and faster alternative for biosynthesis. The simulated dynamic behavior in time of a molecular system is a straight forward source of information about substrate-enzyme interactions at the atomic level, and a powerful tool for the identification of molecular properties important in enzymatic reactions. Our study is focused on the computational investigation of structure and substrate specificity of hydrolases important in biotransformation. The computational work was performed in close collaboration with biochemists-experimentalists from Charles University and the Microbiological Institute of the Academy of Sciences of the Czech Republic. Hydrolases have great a potential in the chemoenzymatic synthesis of modified carbohydrates with regulated properties. Carbohydrates, as substrates of hydrolases, are important in normal functionality of many organisms. They have a dual role in immune response regulation: some carbohydrates (like GlcNAc and ManNAc) participate in activation and some (like GalNAc) in suppressing immunity; glycosidase deficiency is associated with a number of lysosomal disorders. We used homology modeling, computational docking and molecular dynamics simulation (MD) methods for the complex study of fungal hydrolases: alpha-galactosidase/alpha-N-acetylgalactosaminidase from Aspergillus niger; beta-N-acetylhexosaminidases (HEX) (from Aspergillus oryzae and Penicillium oxalicum); nitrilase from Aspergillus niger. Our structural study unambigously demonstrates that the enzyme encoded by genes variant A (aglA) from A. niger is able to accept alpha-N-acetylgalactosamine as its substrate and explains structural features responsible for its specificity. Homology models of HEXs from P. oxalicum and A. oryzae were built and compared. Homology models were used to study the role of protein glycosylation, disulfide bonds, dimer formation and interaction with natural and modified substrates. Model of nitrilase from Aspergillus niger helped to analyze multimer formation.

博士
國立清華大學
化學系
92
Nature has developed many strategies for regulation of enzyme activities, such as phosphorylation and acylation of enzyme molecules themselves, binding of regulatory proteins or molecules (activators, suppressors, or products) at regulatory sites, proteolytic activation of zymogens, etc. In the study of xanthine oxidase catalysis, an unprecedented phenomenon was observed that enzyme substrates themselves could regulate their own or other substrates’ binding affinity and catalytic rates through binding at the active sites of enzyme. Many novel features exist in such a substrate-regulated enzyme system. For example, active sites can also serve as regulatory sites. Substrates can also serve as either activators or suppressors. It is believed that the unprecedented pattern of substrate-regulation of enzyme activities is a general regulation strategy adapted by nature for enzymes with multiple homo-/ hetero- catalytic subunits. A cooperative interaction model was proposed to well describe both positive and negative cooperative effects. The well-known Michaelis-Menten and the Koshland’s “half-of-the-sites” activity models can be treated as special cases of this cooperative catalytic model. Xanthine oxidase (XOD) consists of two identical subunits. For the past 50 years or so, it has been believed that the two subunits carry out catalysis independently. Herein, we report that the presence of 6-formylpterin (6FP), or another substrate, at one of the two active sites affects the binding affinity and the catalytic rate of 6FP at the other active site. These cooperative effects demonstrate unambiguously that the catalytic behavior of the two XOD subunits is strongly interrelated. The inhibition constant (Ki = 8 pM) for the interaction of 6FP with XOD was measured by a stopped-flow method.

Yeung, Ching Yee.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references (leaves 112-122).
Abstracts in English and Chinese.
Acknowledgments --- p.I
Abstract --- p.II
摘要 --- p.III
Content --- p.IV
Abbreviations and symbols --- p.XI
List of tables and figures --- p.XV
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Acylphosphatase --- p.1
Chapter 1.2 --- Human acylphosphatase --- p.4
Chapter 1.3 --- Hyperthermophilic Pyrococcus horikoshii acylphosphatase --- p.5
Chapter 1.4 --- Human common-type acylphosphatase as a mesophilic homologue of Pyrococcus horikoshii acylphosphatase --- p.8
Chapter 1.5 --- Enzyme-substrate interaction of acylphosphatase --- p.9
Chapter Chapter 2 --- Materials and methods --- p.10
Chapter 2.1 --- Preparation of Escherichia coli competent cells --- p.10
Chapter 2.2 --- SDS-polyacrylamide gel electrophoresis --- p.11
Chapter 2.2.1 --- Preparation of polyacrylamide gel --- p.11
Chapter 2.2.2 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.12
Chapter 2.2.3 --- Staining of protein in polyacrylamide gel by Coommassie Brillant Blue R250 --- p.12
Chapter 2.3 --- Expression and purification of Protein --- p.13
Chapter 2.3.1 --- "General bacterial culture, harvesting and lysis" --- p.13
Chapter 2.3.2 --- Purification of acylphosphatase --- p.14
Chapter 2.3.2.1 --- Ion-exchange chromatography --- p.14
Chapter 2.3.2.2 --- Size excision chromatography --- p.15
Chapter 2.3.3 --- Protein concentration determination --- p.16
Chapter 2.4 --- X-ray crystallography --- p.17
Chapter 2.4.1 --- Crystallization of Hu CT AcP --- p.17
Chapter 2.4.2 --- Model building and structural refinement --- p.18
Chapter 2.4.3 --- Crystallization of Hu CT AcP -substate analogue complex --- p.19
Chapter 2.5 --- Enzymatic Assay --- p.21
Chapter 2.5.1 --- Preparation of benzoyl phosphate --- p.21
Chapter 2.5.2 --- Purity check of the BP synthesized --- p.22
Chapter 2.5.3 --- Determination of kinetic parameters of Hu CT AcP --- p.25
Chapter 2.5.4 --- Determination of Ki value of substrate analogue --- p.27
Chapter 2.6 --- Isothermal titration calorimetry --- p.28
Chapter 2.7 --- Reagents and Buffers --- p.30
Chapter 2.7.1 --- Reagent for competent cell preparation --- p.30
Chapter 2.7.2 --- Media for bacterial culture --- p.31
Chapter 2.7.3 --- Reagent for SDS-PAGE --- p.32
Chapter 2.7.4 --- Buffer for AcP purification --- p.33
Chapter 2.7.5 --- Buffer for enzymatic assay and ITC --- p.33
Chapter Chapter 3 --- Structural determination of human common-type acylphosphatase --- p.34
Chapter 3.1 --- Introduction --- p.34
Chapter 3.2 --- Expression and purification of Hu CT AcP --- p.35
Chapter 3.3 --- Structure of Hu CT AcP was determined by X-ray crystallography --- p.37
Chapter 3.3.1 --- Crystallization of Hu CT AcP --- p.37
Chapter 3.3.2 --- Model building and structural refinement --- p.41
Chapter 3.3.3 --- Hu CT AcP shares a same α/β sandwich fold structure as other AcP --- p.43
Chapter 3.4 --- Discussion --- p.46
Chapter 3.4.1 --- Active site structure of Hu CT AcP is the same as those of bovine CT AcP and Ph AcP --- p.46
Chapter 3.4.2 --- Absence of salt bridge between the active site residue and the C-terminal may contribute to the higher catalytic efficiency of Hu CT AcP --- p.52
Chapter Chapter 4 --- Characterization of interaction between acylphosphatase and substrate analogues --- p.56
Chapter 4.1 --- Introduction --- p.56
Chapter 4.2 --- Selected substrate analogues --- p.57
Chapter 4.3 --- Characterization of AcP-substrate analogue interaction by enzymatic assay --- p.59
Chapter 4.3.1 --- Enzyme kinetics of Hu CT AcP was determined by the continuous optical assay of BP hydrolysis --- p.59
Chapter 4.3.2 --- Substrate analogues were found to be competitive inhibitor to the AcP-catalyzed BP hydrolysis --- p.61
Chapter 4.3.3 --- S-BA was the best competitive inhibitor against AcP-catalyzed BP hydrolysis --- p.64
Chapter 4.3.4 --- S-BA was shown to be a competitive inhibitor for both Hu CT and Ph AcP --- p.66
Chapter 4.4 --- Characterization of AcP-substrate analogue interaction by thermodynamic study --- p.68
Chapter 4.4.1 --- Enthalpy change was observed for the association between substrate analogue and AcP --- p.68
Chapter 4.4.2 --- S-BA was shown to bind Hu CT AcP with high affinity in ITC study --- p.68
Chapter 4.5 --- S-BA was found to be the best substrate analogue for AcP --- p.72
Chapter 4.6 --- Discussion --- p.73
Chapter 4.6.1 --- Structure-affinity study of substrate analogue reveals chemical structures essential to interaction with AcP --- p.73
Chapter 4.6.2 --- Structure-affinity study of substrate analogues is consistent with docking model of AcP with acetyl phosphate --- p.75
Chapter 4.6.3 --- Validation of docking model by crystal complex structure --- p.78
Chapter 4.6.4 --- Structural basis of substrate inhibition in Hu CT AcP --- p.80
Chapter 4.6.4.1 --- Substrate inhibition is observed in Hu CT AcP --- p.80
Chapter 4.6.4.2 --- Non-productive binding and substrate inhibition in AcP --- p.80
Chapter Chapter 5 --- Investigation on the effect of salt bridge on acylphosphatase- substrate analogue interaction --- p.84
Chapter 5.1 --- Introduction --- p.84
Chapter 5.2 --- Thermodynamic study on the binding of S-BA with AcPs --- p.87
Chapter 5.2.1 --- Determination of thermodynamic parameters of interaction between AcP and substrate analogue --- p.87
Chapter 5.2.2 --- Determination of thermodynamic parameters as a function of temperature --- p.90
Chapter 5.3 --- Discussion --- p.93
Chapter 5.3.1 --- The presence of salt bridge leads to a reduced flexibility at the substrate binding active site --- p.93
Chapter 5.3.2 --- The single salt bridge reduces the flexibility of active site in both study on thermodynamics of binding and thermodynamics of activation --- p.94
Chapter 5.3.3 --- Temperature dependence of the thermodynamic parameters and heat capacity change ΔCp --- p.97
Chapter 5.3.3.1 --- Change in heat capacity reveals the nature of the complex interface --- p.97
Chapter 5.3.3.2 --- Determination of heat capacity change ΔCp --- p.98
Chapter Chapter 6 --- Structural determination of acylphosphatase-substrate analogue complex --- p.102
Chapter 6.1 --- Introduction --- p.102
Chapter 6.2 --- Soaking and cocrystallization failed to give cocrystal structure of Hu CT AcP and S-BA --- p.103
Chapter 6.4 --- Discussion --- p.106
Chapter 6.4.1 --- Hu CT AcP and S-BA is not compatible with cocrystal formation --- p.106
Chapter 6.5 --- Future prospect --- p.107
Chapter 6.5.1 --- Structure determination by NMR spectroscopy --- p.107
Chapter 6.5.2 --- Structure determination of AcP with aluminofluoride complexes --- p.108
Chapter Chapter 7 --- Conclusion --- p.109
Reference --- p.112

N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs.

Mass spectrometry has enjoyed enormous popularity over the years for studying biological systems. The theme of this dissertation was to develop and use mass spectrometry based tools to solve five biologically oriented problems associated with protein architecture and extend the utility of these tools to study protein polymer conjugation. The first problem involved elucidating the false negatives of how proteins with few basic residues, forms highly charged ions in electrospray ionization mass spectrometry (ESI MS). This study showed that the unfolding of polypeptide chains in solution leads to the emergence of highly charged protein ions in ESI MS mass spectra, even if the polypeptide chains lack a sufficient number of basic sites. In the second problem, a new technique was developed that can monitor small-scale conformational transitions that triggers protein activity and inactivity using porcine pepsin as a model protein. This work allowed us to revise a commonly accepted scenario of pepsin inactivation and denaturation. The physiological relevance of an enzyme-substrate complex was probed in our third problem. We observed by ESI MS that pepsin forms a facile complex with a substrate protein, N-lobe transferrin under mildly acidic pH. The observed complex could either be a true enzyme-substrate complex or may likely results from an electrostatically driven association. Our investigation suggested that the enzyme binds nonspecifically to substrate proteins under mild acidic pH conditions. The fourth problem dealt with the investigation of conformational heterogeneity of natively unstructured proteins using a combination of spectroscopic techniques and ESI MS as tools. It was observed that four different conformations of alpha-synuclein coexist in equilibrium. One of these conformations appeared to be tightly folded. Conclusions regarding the nature of these states were made by correlating the abundance evolution of the conformers as a function of pH with earlier spectroscopic measurements. The final problem was aimed at monitoring conformational transitions in polypeptide and polymer segments of PEGylated proteins using PEGylated ubiquitin as a model system. This studies suggested that for a PEGylated protein, polypeptides maintain their folded conformation to a greater extent whiles the polymer segments are bound freely to the protein.