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Dissertations / Theses on the topic 'Substrate specificity'

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Published: 4 June 2021
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1

Babcock, Gwen. "Maize β-glucosidase substrate specificity and natural substrates." Thesis, Virginia Tech, 1993. http://hdl.handle.net/10919/45360.

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2

Babcock, Gwen. "Maize [beta]-glucosidase substrate specificity and natural substrates /." This resource online, 1993. http://scholar.lib.vt.edu/theses/available/etd-10312009-020235/.

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3

Allison, Timothy Murray. "Substrate specificity and mutational studies of KDO8PS." Thesis, University of Canterbury. Chemistry, 2012. http://hdl.handle.net/10092/6684.

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The enzyme 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyses the stereospecific aldol-like condensation between phosphoenolpyruvate (PEP) and the five-carbon sugar D-arabinose 5-phosphate (A5P). This is the first biosynthetic step in the formation of 3-deoxy-D-manno-octulosonate (KDO), an essential lipopolysaccharide component of all Gram-negative bacteria. KDO8PS is evolutionarily related to the shikimate pathway enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), which catalyses a similar condensation reaction between PEP and the four-carbon sugar D-erythrose 4-phosphate (E4P), in the first step of the shikimate pathway to aromatic compounds in plants and microorganisms. As well as being a one-carbon shorter substrate, E4P has the opposite C2-OH configuration to A5P. While there are both metal-dependent and metal-independent forms of KDO8PS, in contrast, all DAH7PS are metal-dependent enzymes. Little is understood about the key sequence features that distinguish KDO8PS and DAH7PS. These features, particularly those that contribute to A5P or E4P binding, are thought to be responsible for the differences in substrate specificity between the two enzymes. This thesis describes the functional and structural studies of KDO8PS mutants to examine the roles of these residues, using the metal-dependent KDO8PS from Acidithiobacillus ferrooxidans and the metal-independent KDO8PS from Neisseria meningitidis. In Chapter 2 an extensive KDO8PS and DAH7PS sequence analysis is presented. The results, which identify sequence conservation in both enzymes, are discussed in the context of the (β/α)8 TIM-barrel structure. Some of the differences in conservation between the two enzymes were highlighted as being obvious in having a role or contributing to the different substrate selection preferences of the two enzymes, such as an extended β7α7 loop in KDO8PS, and motif differences on the β2α2 and β4α4 loops. A similar analysis was also used to compare metal-dependent and metal-independent KDO8PSs, and it was found the two forms differ in the conservation of only three residues. Chapter 3 describes the characterisation of A. ferrooxidans KDO8PS (AfeKDO8PS) and investigates aspects of metal dependency in KDO8PS. The enzyme was found to be metal dependent, and like all other KDO8PS enzymes, to possess a tetrameric quaternary structure, and display tight substrate specificity. The β8α8 loop was found to have a critical role in binding and positioning the substrates, and AfeKDO8PS could not be engineered to be a metal-independent enzyme. The role of the KDO8PS-conserved KANRS motif, present on the β2α2 loop and one of the main contributors to the A5P binding site, is probed in Chapter 4. Individual residues of the motif were mutated to investigate function, and the motif was converted to the equivalent motif found in DAH7PS (KPRS). It was found that the Lys plays a critical role in enzymatic catalysis, and is likely intimately involved in the enzyme mechanism. The Asn residue of the motif in KDO8PS was found to be an important contributor to KDO8PS stereospecificity. The work described in Chapter 5 investigates the role of the β7α7 loop in KDO8PS. This long active-site loop, which exists in a shorter version in DAH7PS, was found not to be essential for catalysis in KDO8PS, but was necessary for efficient catalysis. The two conserved residues on the loop provide interactions to A5P, but the presence of the extended loop as a whole was found to be most important for catalytic efficiency. In Chapter 6 a conserved residue on the re face of PEP is investigated. In KDO8PS the residue is conserved as Asp, and in DAH7PS the same residue is conserved as a Glu. Mutational analysis found that in KDO8PS the Asp residue appears to be important for enzyme activity but unimportant for PEP binding. Mutating this Asp in KDO8PS to Glu was accommodated by KDO8PS, but it was found its introduction could potentially be optimised by coupling the change with mutation to other conserved differences. In KDO8PS, one of the interfaces between adjacent subunits in the tetrameric structure is partially composed of a conserved sequence motif, PAFLxR. In Chapter 7, the roles of the residues in this motif are explored. The Arg of the motif was found to be important for A5P binding. The equivalent (and also conserved) motif in DAH7PS is GARNxQ, and mutation of residues in the KDO8PS motif to the equivalent residues in DAH7PS was tolerated by KDO8PS, but negatively impacted upon the enzyme kinetic parameters. The sequence features investigated in the other chapters were combined with those to the subunit interface to create a DAH7PS-like protein. This extensively engineered protein lost all KDO8PS activity, but nor did it gain DAH7PS activity. Lastly, in Chapter 8 the results from all chapters are reviewed and ideas are discussed for advancing the research presented in this thesis.
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4

Chappell, Lucy. "Engineering the substrate specificity of galactose oxidase." Thesis, University of Leeds, 2013. http://etheses.whiterose.ac.uk/5741/.

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Biocatalysis, the use of enzymes to catalyse the generation of specific chemicals, has a number of advantages including high specificity, lower energy requirements and greater sustainability over an equivalent chemical process. Several methods exist for optimisation of enzymes for use in industrial processes, including introduction of mutations to generate libraries of variants which are then screened for the desired properties, such as stability or substrate specificity. Galactose oxidase catalyses the oxidation of primary alcohols to the corresponding aldehyde, with concomitant reduction of dioxygen to hydrogen peroxide. It has already been developed for use in a range of biotechnological processes and is an ideal candidate for further development due to features including high stability, a surface exposed active site displaying broad substrate specificity, and an autocatalytically-generated cofactor. Research presented in this thesis investigates the effect on activity towards a range of alternative substrates of mutations at selected active site residues with the aim of expanding the biotechnological potential of galactose oxidase. Libraries of variants were designed and generated using high quality oligonucleotides constructed using trimer phosphoramidites. Screening assays used by other groups were optimised by varying different components. These assays were then used to identify a number of variants displaying enhanced activity towards D-arabinose, D-glucose, D-xylose or glycerol. A selection of these variants were then further characterised in order to understand the biochemical basis of the altered activities and determine some of the conditions required for potential industrial application of the variants. The most exciting results include identification of a variant displaying higher levels of activity towards glycerol than towards the native substrate D-galactose; determination of the position of oxidation of D-arabinose at the C-4 hydroxyl; and the observation that mutation of Phe194 significantly affects binding of D-glucose in the active site.
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5

Ahn, Jinwoo. "DNA polymerase ? : Control of substrate specificity and fidelity /." The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487943610785207.

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6

Björnberg, Olof. "Viral dUTPases recombinant expression, purification, and substrate specificity /." [Lund] : Dept. of Biochemistry, Lund University, Sweden, 1995. http://books.google.com/books?id=hvZqAAAAMAAJ.

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7

Lee, Nicholas Yong Kyu. "Characterizing substrate specificity and affinity in zebrafish deiodinases." Thesis, Boston University, 2012. https://hdl.handle.net/2144/12472.

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Thesis (M.A.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Thyroid hormones are important for development and growth and their metabolism is mediated by a special class of enzymes called deiodinases. In this study, we cloned zebrafish deiodinases 1-3 (sequences from GenBank) and transfected them into mammalian cells. A special sequence called the selenocysteine insertion sequence was also cloned and transfected to express zebrafish deiodinases at high levels. Deiodination activity from the cloned zebrafish deiodinases indicated that GenBank sequences encode functional enzymes with the same specificity as human deiodinases. Zebrafish D1 was highly effective in catalyzing the outer ring deiodination of rT3. Zebrafish D2 catalyzed the outer ring deiodination of all tested substrates but showed no inner ring deiodination activity. Zebrafish D3 only catalyzed the inner ring deiodination of T3 into T2. We also observed that all zebrafish deiodinases required the SECIS element for enzyme activity. Furthermore, we demonstrated that the optimal temperature for zebrafish D3 catalyzed T3 deiodination is at room temperature instead of previously thought 28.5° C. The dramatic difference in zebrafish D3 (23.0° C compared to human D3 at 37.0° C) illustrated that there is an important difference between species. Finally, we demonstrated that zebrafish D3 has high affinity for T3 through Lineweaver Burk analysis and showed that the Km value of zebrafish D3 is in the low nanomolar range similar to human D3. Together with high substrate specificity for T3, we demonstrated that"zebrafish D3 is the primary inactiviator of T3 in zebrafish. We concluded that zebrafish deiodinase sequences in GenBank encode functional enzymes with high affinity and specificity but require the presence of the SECIS element for enzyme activity. Furthermore, we concluded that there is an important difference in optimal temperature between mammalian and zebrafish D3.
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8

Townsend, Andrew Paul. "Nitrogen mustards as tools in determining methyltransferase substrate specificity." Thesis, University of Nottingham, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.517857.

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9

Bolt, Amanda Helen. "Probing the substrate specificity and stereoselectivity of an aldolase." Thesis, University of Leeds, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.507677.

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10

Cohen, H. "Investigating and engineering the substrate specificity of DNA methyltransferases." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597811.

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DNA methyltransferases catalyse the transfer of methyl groups from the cofactor S-adenosyl-L-methionine to a target base (adenine or cytosine) within a cognate recognition sequence. I have studied cofactor binding, DNA specificity and the role of conserved amino acid motifs in the cytosine C5 methyltransferase M.HaeIII. By measuring the competitive inhibition of methylation by a series of cofactor analogues, each modified at a single position, the importance of each functional group for cofactor binding to M.HaeIII was probed. The functional significance of amino-acid residues in M.HaeIII was investigated using in vitro compartmentalisation (IVC), an activity-based selection method. IVC was used to obtain active variants of M.HaeIII from libraries diversified at conserved motifs in the catalytic and DNA binding domains. M.HaeIII modifies the central cytosine of the sequence (5’-GGCC-3’). Using bisulphite sequencing, cytosines in a variety of other sequence contexts were found to be methylated at lower levels by M.HaeIII both in vivo and in vitro. IVC was then used to select mutant enzymes with an improved ability to methylate the non-canonical site AGCC.
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11

Carlson, Jacob Charles. "Continuous Directed Evolution of Enzymes with Novel Substrate Specificity." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11053.

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Methodological advances in directed evolution have already made it possible to discover useful biomolecules within months to years. A further acceleration of this process might make it possible to address outstanding challenges, or needs for which the current timescale is a fundamental barrier. To realize these goals would require transformative methodological advances in directed evolution. In Chapter One, current methods in directed evolution are briefly reviewed. In Chapter Two, a general platform for continuous directed evolution is presented. The method is used to evolve T7 RNA polymerase enzymes with novel promoter activity on the days timescale. In Chapter Three, a method is developed for tuning selection stringency during continuous evolution, thus obviating the requirement for a minimal starting library activity. In Chapter Four, a method is developed for simultaneous positive and negative selection, thus allowing explicit selection for substrate specific enzymes. In Chapter Five, the advances in stringency modulation and negative selection are combined to evolve highly substrate specific enzymes starting from an inactive starting library. In a continuous evolutionary arc of less than three days, we discover T7 RNA polymerase enzymes with a degree of specificity for the T3 promoter exceeding that of the wild type enzyme for its native substrate.
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12

Penfield, Jonathan. "The substrate specificity and conformational flexibility of ketosteroid hydroxylases." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/45138.

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3-Ketosteroid-9α-hydroxylase (KshAB) is a Rieske oxygenase involved in bacterial steroid degradation. Bacteria such as Rhodococcus rhodochrous DSM43269 harbor up to five KshA homologues (numbered 1 to 5) that appear to be involved in degrading different steroids. Previous work indicated that KshA5 (DSM43269) transforms an unusually broad range of 3-ketosteroids while KshA1 (DSM43269) and KshAMtb of Mycobacterium tuberculosis have strong preference for 3-ketosteroids with side chains at C-17. To better understand KshAs in general, KshA1 and KshA5 were purified anaerobically and characterized. Steady-state kinetic studies revealed that KshA1 has 10- to 100-fold higher apparent specificity constant for ketosteroids possessing long C17 side chain such as 3-oxo-23,24-bisnorchola-1,4-dien-22-oate (4-BNC), and is thus similar to KshAMtb. By contrast, KshA5 had highest specificity for substrates with C17-oxo (e.g., apparent kcat/Km > 10⁵ s-¹ M-¹ for 4-estrendione and 5α-androstandione vs. 10² s-¹ M-¹ for 1,4-BNC-CoA). However, KshA5 displayed very strong substrate inhibition with 1,4-androstadiene-3,17-dione (ADD) and 4-BNC (KSS ~110 μM) despite hydroxylation well coupled to O₂ consumption and turnover occurring at reasonable rates (apparent kcat ~0.7 s-¹). Crystallographic structures of four KshA:substrate complexes were determined: KshA1:ADD (2.4 Å), KshAMtb:ADD (2.3 Å), KshA5:ADD (1.8 Å) and KshA5:1,4-BNC-CoA (2.6 Å). In each, the substrate was bound in a similar orientation with the steroid’s C9 closest to the active site iron. In comparison to a structure of substrate-free KshA5 (2.6 Å resolution), the catalytic iron was displaced up to 3.1 Å in the complexes with ADD and 1,4-BNC-CoA. This was accompanied by similar magnitude shift in the helices harboring the iron-coordinating residues. The net effect was an unusually large distance between the iron and C9 of the substrate (6.3 Å). Additionally, the active site opening of KshA5 was occluded from bulk solvent by a loop comprising residues 217 to 233 in substrate-free and ADD-bound structures while the KshA5:1,4-BNC-CoA complex exhibited an open active site, as observed in KshA1 and KshAMtb structures, containing a similar disordered loop region. The loop conformation in these structures and the ability of KshA5 to turnover CoA thioesters demonstrate unexpected conformational flexibility in correlation with interesting kinetic behavior in a Rieske oxygenase.
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13

Bagneris, Claire. "Protein engineering of benzene dioxygenase for altered substrate specificity." Thesis, King's College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326106.

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14

Stein, Benjamin J. (Benjamin Joseph). "Substrate specificity of [alpha]-proteobacterial N-end rule adaptors." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/104102.

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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis. "June 2016." In title on title page [alpha] appears as lower case Greek letters.
Includes bibliographical references (pages 103-118).
by Benjamin J. Stein.
Ph. D.
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15

Yerkes, Nancy (Nancy Mary). "Purification and substrate specificity of new C. roseus enzymes." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/62061.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2010.
Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
Terpene indole alkaloids (TIAs) are a class of natural products produced in plants. Many TIAs have medicinal uses; for example, vinblastine has anti-cancer activity and ajmaline has anti-arrhythmic activity. Many TIAs did not evolve to treat human disease, however, and thus most likely do not have optimal pharmacological properties. If TIAs could be modified, the novel TIAs produced could have improved bioactivities when compared with the unmodified natural TIAs. Unfortunately, the immense structural complexity of TIAs makes cost-effective industrial-scale synthesis of the majority of TIAs and TIA analogs unfeasible. Industrial-scale production of TIAs would be improved if TIAs could be produced via reconstitution of the enzymatic pathways in a heterologous organism such as yeast. However, many of the enzymes involved in TIA biosynthesis are unknown, thereby precluding these efforts. If more TIA biosynthetic enzymes were isolated, and the substrate specificity of the enzymes were known, both natural and novel TIA analogs could be more readily produced on an industrial scale. In this thesis I developed strategies to isolate new C. roseus enzymes and to make novel analogs of the anti-hypertensive agent ajmalicine and the anti-neoplastic agent isositsirikine. The NADPH-dependent reductases that produce ajmalicine and isositsirikine have not been isolated. To produce ajmalicine and isositsirikine analogs in vitro, two aims must be accomplished: first, the reductases forming ajmalicine and isositsirikine, ajmalicine synthase and isositsirikine synthase, must be partially purified, and second, the substrate specificity of those reductases must be determined. To satisfy the first of these aims, I developed a partial purification procedure for ajmalicine synthase and isositsirikine synthase from Catharanthus roseus tissue. My partial purification procedure involved acetone precipitation, ion exchange chromatography, and gel filtration chromatography. Analysis by 2D SDS-PAGE shows that the proteins have been significantly purified. I also performed crosslinking experiments with a substrate probe in attempts to isolate ajmalicine synthase and isositsirikine synthase. In the crosslinking studies four enzymes were isolated and cloned, and one has been found to have sinapyl alcohol dehydrogenase activity. I determined the substrate specificities of ajmalicine synthase and isositsirikine synthase' as well as the enzyme that precedes both enzymes in the biosynthetic pathway, strictosidine-pglucosidase (SGD). I found that SGD, ajmalicine synthase, and isositsirikine synthase all have broad substrate specificities, which is promising for the development of novel ajmalicine and isositsirikine analogs with potentially improved therapeutic activities.
by Nancy Yerkes.
Ph.D.
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16

Howell, Nathan W. "Substrate specificity of the Trm10 m1R9 tRNA methyltransferase family." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1563209805137069.

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17

Sharpe, Amy-Joan Lorna 1965. "Substrate specificity of rat liver aldehyde dehydrogenase with chloroacetaldehydes." Thesis, The University of Arizona, 1991. http://hdl.handle.net/10150/277906.

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Chlorinated acetaldehydes have recently been the focus of research due to their role as reactive intermediates and their possible occurrence in chlorinated drinking water. The metabolism of these compounds, however, has not been extensively studied. In this study, the in vitro substrate specificity of cytosolic and mitochondrial rat liver aldehyde dehydrogenase toward these compounds was investigated. Both crude and semi-purified preparations of the enzymes were used. Monochloroacetaldehyde was found to be extensively metabolized by this enzyme system. It was metabolized to a greater extent than the standard compound propionaldehyde. Dichloroacetaldehyde was also found to be metabolized by this enzyme, but to a lesser extent than its monochloro-analogue. There was some evidence to suggest, however, that alcohol dehydrogenase and chloral hydrate dehydrogenase may play a significant role in the metabolism of this compound. Chloral hydrate was not metabolized by this enzyme to an appreciable extent.
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18

MacMillan, Susan Veronica. "The substrate and inhibitor specificity of the osmoregulatory transporter ProP." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ33248.pdf.

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19

Hancock, Susan M. "Engineering the substrate specificity and mechanism of a thermophilic glycosidase." Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414143.

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20

Hill, Cheryl Louise. "Investigation into the substrate specificity of 6-oxo camphor hydrolase." Thesis, University of York, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.440690.

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21

Farrell, Christopher Mark. "Specificity and regulation of substrate degradation for a AAA+ protease." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/38998.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007.
Includes bibliographical references.
Energy dependent proteolysis is a critical method of cellular regulation for all forms of life. The AAA+ proteases ClpXP and ClpAP in E. coli function in this capacity by facilitating the denaturation and degradation of target substrates. These proteolytic enzymes degrade hundreds of different proteins. Determining how the activities of these proteases are regulated in the cell as well as learning how these enzymes bind and engage substrates are important goals. In order to better understand how the degradation of ClpXP and ClpAP is regulated, I studied their contributions to ssrA-tagged protein degradation in the cell. Using GFP-ssrA expressed from the chromosome as a degradation reporter, the effects of altered concentrations of different protease components or adaptor proteins were explored. I found that both ClpXP and ClpAP could degrade GFP-ssrA in the cell and that increased levels of ClpAP in stationary phase resulted in increased degradation of ssrA-tagged substrates. I also demonstrated that wild-type levels of the adaptor proteins SspB and ClpS do not fully inhibit ClpAP degradation of GFP-ssrA. To better understand how the ClpXP enzyme binds substrates, I took a mutagenic approach.
(cont.) The "RKH" loops surround the entrance to the central pore of the ClpX hexamer and are highly conserved in the ClpX subfamily of AAA+ ATPases. I discovered that a mutation within the RKH loop of ClpX changes substrate specificity by 300-fold, resulting in decreased degradation of ssrA-tagged substrates but improved degradation of proteins with other classes of degradation signals. My results show that the RKH loops recognize the C-terminal carboxylate of the ssrA tag and suggest that ClpX specificity represents an evolutionary compromise that has optimized degradation of multiple types of substrates rather than any single class.
by Christopher Mark Farrell.
Ph.D.
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22

Harsch, Christina I. K. "Mutagenic Studies of Substrate Specificity and Stability of Paraoxonase-1." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1325197473.

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23

dela, Seña Carlo C. "Substrate specificity and reaction mechanism of vertebrate carotenoid cleavage oxygenases." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1396444100.

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24

Hariharan, Balasubramani. "Structure-Function and Substrate-Specificity Studies of Escherichia coli YidC." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1534461559986884.

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25

Blewett, Anne Morwenna. "The substrate specificity of peptidoglycan biosynthesis enzymes from Streptococcus pneumoniae." Thesis, University of Warwick, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422083.

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26

Bartels, Kim [Verfasser]. "Conformational dynamics and substrate specificity in nutrient transporters / Kim Bartels." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2020. http://d-nb.info/122504197X/34.

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27

Zhang, Suyang. "Mechanism of APC/C activation and substrate specificity in mitosis." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275479.

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In eukaryotes, cell proliferation and cell cycle transitions are strictly controlled by the anaphase-promoting complex/cyclosome (APC/C). The APC/C is an E3 ubiquitin ligase that regulates chromatid segregation at the metaphase to anaphase transition, exit from mitosis and the establishment and maintenance of G1. The APC/C’s catalytic activity and substrate specificity are controlled by its interactions with two coactivators, Cdc20 and Cdh1. In contrast to Cdh1, APC/C activation by Cdc20 during mitosis requires hyper-phosphorylation of APC/C subunits by cyclin-dependent kinase (Cdk) and polo kinase. The aim of the first part of this thesis was to understand how mitotic phosphorylation regulates APC/C activity. Using cryo-electron microscopy and biochemical analysis, we found that an auto-inhibitory segment of the Apc1 subunit acts as a molecular switch that in apo unphosphorylated APC/C interacts with a coactivator-binding site (C-box binding site), thereby obstructing engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box binding site to relieve APC/C auto-inhibition. Efficient phosphorylation of the auto-inhibitory segment requires the recruitment of the kinase Cdk-cyclin-Cks to a hyper-phosphorylated loop of Apc3. In addition to regulation of APC/C activity by phosphorylation, ordered cell progression is ensured by the ability of the APC/C to target substrate degradation in a defined order. At mitosis onset, degradation of securin and cyclin B1 is inhibited by the spindle assembly checkpoint, exerted by the mitotic checkpoint complex (MCC), whereas both cyclin A2 and Nek2A are not subject to MCC inhibition. The aim of the second part of the thesis was to elucidate the mechanism of how the APC/C achieves its substrate specificity. Our biochemical analysis showed that the resistance of cyclin A2 to MCC inhibition is due to its ABBA motif and the Cdk-associated Cks2 subunit. Furthermore, we found that it is the Cdc20 molecule of the MCC that binds to the ABBA motif to allow for cyclin A2 ubiquitination. Strikingly, mutating all three known degrons (KEN box, D box and ABBA motif) of cyclin A did not affect its ubiquitination by APC/CCdc20. Deletion of a potential novel degron found within residues 60-80 of cyclin A2 impaired cyclin A2 ubiquitination.
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28

Mörtl, Mario Samuel. "Substrate specificity of Glycine Oxidase and protein interaction specificity of the neuronal cell adhesion molecule TAG-1." [S.l. : s.n.], 2006. http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-66181.

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29

Fransson, Linda. "Enzyme substrate solvent interactions : a case study on serine hydrolases." Doctoral thesis, KTH, Biokemi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4867.

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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
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30

Hart, K. W. "An investigation into the molecular basis of substrate specificity in lactate dehydrogenase." Thesis, University of Bristol, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.235201.

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31

Morden, Darrell Shawn. "Development of a selection system for proteases with novel substrate specificity." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ61593.pdf.

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32

Boerboom, Derek. "Subcellular localization and substrate specificity of the protein-tyrosine phosphatase MPTP." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=22850.

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In order to study the subcellular localization of the protein-tyrosine phosphatase MPTP, indirect immunofluorescence microscopy experiments were performed. The enzyme was thereby determined to localize almost exclusively to the nucleus. The nuclear localization signal was mapped by further immunofluorescence experiments performed with deletion mutants and $ beta$-galactosidase fusion constructs. The sequence $ rm sp{345}RKRIRED sp{351}$ was found to be capable and sufficient to localize MPTP and unrelated proteins to the nucleus, and a secondary domain was identified that is likely involved in mediating the nuclear retention of MPTP. To determine the in vivo functions of MPTP, its substrate specificity was analyzed. Dephosphorylation assays of cellular proteins identified a preferred substrate of an approximate molecular weight of 40 kiloDaltons (kDa) and an isoelectric point (pI) of 4.9. This and another substrate of approximately 43kDa and a pI of 4.8 were identified in a trapping assay using a catalytically inert mutant of MPTP as an affinity reagent, and could be isolated from two independent cell lines. Taken together, these data provide significant advances towards the identification of the signal transduction pathways in which MPTP is involved.
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33

Hunter, Michael Forbes Clifford. "Controlling the substrate specificity of α-isopropylmalate synthase and related enzymes." Thesis, University of Canterbury. Department of Chemistry, 2013. http://hdl.handle.net/10092/8727.

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The enzyme α-isopropylmalate synthase (IPMS) catalyses the reaction between acetyl coenzyme A (AcCoA) and α-ketoisovalerate (KIV) to produce free coenzyme A and α isopropylmalate (IPM). This reaction is a key control point in the biosynthesis of a leucine, a pathway absent in animals but present in plants, fungi and bacteria. As a result, IPMS is a antibiotic and herbicidal target that has been validated by knockout studies for M. tuberculosis, the causative agent of tuberculosis. Engineered IPMSs have also been used in the fermentative production of long chain alcohols for use as fuels. IPMS belongs to a family of related enzymes called α-ketoacid: AcCoA re-aldolases (KARAs), with each subfamily differing in the specific α-ketoacid that AcCoA is reacted with. The known KARA subfamilies are IPMS, citramalate synthases (CMSs), homocitrate synthases (HCSs), methylthioalkylmalate synthases (MAMSs) and re-citrate synthases (RCSs), respectively involved in the biosynthesis of isoleucine, lysine, glucosinolates and TCA cycle intermediates. This thesis describes work aimed at improving understanding of both specific subfamilies of KARA enzymes and also the genetic and functional relationships between the subfamilies. A particular emphasis is placed on relating primary structure to function, allowing the inference of function from a very small subset of residues. IPMSs are divided into two classes, the Mtu-like IPMSs and the much less studied Eco-like IPMSs. Chapter 2 details the expression and characterisation of the Eco like IPMS from N. meningitidis (NmeIPMS). Overall NmeIPMS showed similar properties to MtuIPMS, but unlike that enzyme NmeIPMS is inhibited by high divalent metal ion concentrations, does not require monovalent metal ions, and shows some activity with the α-ketoacid 3-methyl α ketovalerate. Several previous results showing inhibitory activity of Zn2+, Cd2+ and bromopyruvate were also found to be the results of interference with the assay system and all three were found to be much weaker inhibitors than previously determined.   Phylogenetic analysis of the different KARA subfamilies revealed certain specific positions that were believed to control substrate specificity. Chapter 3 details mutagenesis experiments on MtuIPMS that probe the function of these residues. Once the importance of the residues had been established, substitutions were made in which IPMS residues were replaced with their equivalents from HCSs and CMSs in order to change substrate specificity. The most successful result was the Y169L substitution based on HCS, which decreased the specificity constant with KIV by four orders of magnitude while improving other activities, successfully shifting the best activity to the unbranched α-ketoacid α-ketobutyrate. Chapter 4 of this thesis details the purification and functional testing of the RCS from C. saccharolyticus (CscRCS), the first thermophilic RCS characterised. CscRCS was found to have an extremely low Km for its substrate oxaloacetate (1.7 µM), believed to be an adaptation to the instability of oxaloacetate at the temperatures CscRCS operates at in vivo. The enzyme also showed competitive affinity by α-ketoglutarate, the end product of the pathway. Unlike other characterised RCSs, CscRCS showed no oxygen sensitivity. The phylogenetic analysis conducted for this thesis identified a subfamily of KARAs dubbed pseudo-IPMSs (PIPMSs) that showed no substantial homology to any studied subfamily. In Chapter 5 the PIPMS from T. thermophilus (TthPIPMS) was expressed and characterised. TthPIPMS showed many features of a CMS, being most active with the same substrate (pyruvate) and sensitive to the same inhibitor (isoleucine). Unlike the previously studied CMS subfamilies, TthPIPMS possesses a nanomolar IC50 for its inhibitor, and also shows substantial activity as an RCS. The results of these chapters are then drawn together in Chapter 6 to create a picture of the relationships between the KARA enzymes, in terms of their functional characteristics as well as the sequence and evolutionary relationships between them that have bought about their diverse functions.
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34

Debenham, Donna Michelle. "Characterisation of MTMR3 : a phosphatidylinositol 3 phosphatase with novel substrate specificity." Thesis, University of Liverpool, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272602.

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35

Chen, Mark M. "Investigating asparagine-linked glycosylation substrate : specificity and effects on protein folding." Thesis, Massachusetts Institute of Technology, 2009. http://hdl.handle.net/1721.1/46644.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009.
Vita.
Includes bibliographical references.
N-linked glycosylation is a ubiquitous form of protein modification whereby a preassembled oligosaccharide is covalently attached the asparagine side chain of an acceptor protein. This process involves numerous enzymes, produces a diverse set of oligosaccharide structures, and results in a variety of structural and functional effects on the glycoprotein. Research discussed in this dissertation applies synthetic organic chemistry to probe this important biological system. To study the effects of N-linked glycosylation on protein folding, a semi-synthetic strategy was developed to access a set of model proteins that were homogeneously glycosylated at several sites of interest. The folding kinetics of this set of glycoproteins were then characterized using stopped-flow fluorescence spectroscopy, which revealed that the presence of the glycan show discrete effects on both the rate of protein folding and unfolding, and that the overall effect is highly specific to the local primary and secondary structure of the glycosylation site. The gram-negative bacterium Campylobacterjejuni was recently discovered to contain a general N-linked glycosylation system with a defined glycan structure and tractable enzymes for heterologous expression including a single subunit oligosaccharyltransferase. To probe the bacterial N-linked glycosylation machinery, a chemo-enzymatic synthesis for each of the glycan intermediates within this pathway was developed, which are impractical to obtain from the host organism. Importantly, chemo-enzymatic allowed for the incorporation of structural modifications for binding-specificity assays and radiolabels for accurate quantification. Access to these substrates allowed us to define the minimum glycosylation consensus sequence for the oligosaccharyltransferase as well as the polyisoprenol specificity of three representative enzymes within the pathway.
by Mark M. Chen.
Ph.D.
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36

Keiler, Kenneth Charles. "Substrate specificity, active-site residues, and function of the Tsp protease." Thesis, Massachusetts Institute of Technology, 1995. http://hdl.handle.net/1721.1/39746.

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37

Harrison, Jennifer Amanda. "Investigation of the substrate specificity of recombinant Trypanosoma cruzi trans-sialidase." Thesis, University of St Andrews, 1999. http://hdl.handle.net/10023/14907.

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The protozoan blood-borne parasite Trypanosoma cruzi is the causative agent of Chagas' disease, an enervating and often fatal illness prevalent in South and Central America for which there is no effective treatment. T. cruzi has a cell-surface trans- sialidase which transfers sialic acid from mammalian oligosaccharides to the parasite. This action allows adhesion to and invasion of mammalian cells, subsequently allowing parasitic replication. This protein therefore is exploitable and represents a potential target for the development of chemotherapeutic agents. This thesis describes the purification of recombinant trans-sialidase and the development of a rapid, reliable spectrophotometric coupled assay to measure trans-sialidase activity. It also details the use of three mutually exclusive synthetic oligosaccharide libraries to map substrate recognition for the enzyme. Synthetic fragments of the natural branched oligosaccharide substrates have also been sialylated on a preparative scale, demonstrating the use of trans-sialidase in synthetic oligosaccharide chemistry.
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38

Hou, Shurong. "Structural Mechanism of Substrate Specificity In Human Cytidine Deaminase Family APOBEC3s." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1079.

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APOBEC3s (A3s) are a family of human cytidine deaminases that play important roles in both innate immunity and cancer. A3s protect host cells against retroviruses and retrotransposons by deaminating cytosine to uracil on foreign pathogenic genomes. However, when mis-regulated, A3s can cause heterogeneities in host genome and thus promote cancer and the development of therapeutic resistance. The family consists of seven members with either one (A3A, A3C and A3H) or two zinc-binding domains (A3B, A3D, A3D and A3G). Despite overall similarity, A3 proteins have distinct deamination activity and substrate specificity. Over the past years, several crystal and NMR structures of apo A3s and DNA/RNA-bound A3s have been determined. These structures have suggested the importance of the loops around the active site for nucleotide specificity and binding. However, the structural mechanism underlying A3 activity and substrate specificity requires further examination. Using a combination of computational molecular modeling and parallel molecular dynamics (pMD) simulations followed by experimental verifications, I investigated the roles of active site residues and surrounding loops in determining the substrate specificity and RNA versus DNA binding among A3s. Starting with A3B, I revealed the structural basis and gatekeeper residue for DNA binding. I also identified a unique auto-inhibited conformation in A3B that restricts access to the active site and may underlie lower catalytic activity compared to the highly similar A3A. Besides, I investigated the structural mechanism of substrate specificity and ssDNA binding conformation in A3s. I found an interdependence between substrate conformation and specificity. Specifically, the linear DNA conformation helps accommodate CC dinucleotide motif while the U-shaped conformation prefers TC. I also identified the molecular mechanisms of substrate sequence specificity at -1’ and -2’ positions. Characterization of substrate binding to A3A revealed that intra-DNA interactions may be responsible for the specificity in A3A. Finally, I investigated the structural mechanism for exclusion of RNA from A3G catalytic activity using similar methods. Overall, the comprehensive analysis of A3s in this thesis shed light into the structural mechanism of substrate specificity and broaden the understanding of molecular interactions underlying the biological function of these enzymes. These results have implications for designing specific A3 inhibitors as well as base editing systems for gene therapy.
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39

Kimani, Serah. "Catalysis, substrate binding and specificity in the amidase from Nesterenkonia species." Doctoral thesis, University of Cape Town, 2011. http://hdl.handle.net/11427/10837.

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To investigate the structural determinants of NitN specificity on short aliphatic amide substrates by analyzing binding and interactions of these molecules with the NitN binding pocket. To probe the catalytic role of the two active site glutamate residues (Glu61 and Glu139) using NitN as a model enzyme. To monitor the activity, interactions and reactivity of the WT NitN and the Glu61 and Glu139 NitN mutants with ACR.
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40

Kojima, Yuzo. "Screening and applied studies of microbial lipases with unique substrate specificity." Kyoto University, 2006. http://hdl.handle.net/2433/136636.

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41

Wu, Jinzi. "Studies of substrate specificity, regulation and inhibition of protein-tyrosine kinases." Diss., The University of Arizona, 1996. http://hdl.handle.net/10150/282141.

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Protein tyrosine kinases (PTKs) play a crucial role in the regulation of cell proliferation, differentiation and signal transduction. However, substrate specificity and recognition motifs are not clear for most PTKs. Traditional methodologies for identifying substrate recognition motifs are often difficult and inefficient. In this dissertation, a novel approach for rapid discovery of linear substrate motifs of protein kinases has been developed using cyclic AMP-dependent protein kinase (cAPK) as a model system. This method is based on the screening of random synthetic combinatorial peptide libraries on beads where each bead expresses only one peptide entity. The peptide motif identified was RRXS, which exactly matched the motif for cAPK reported in the literature. Using this approach, a fraction of the random peptide library was screened for substrates of c-Src PTK. A heptapeptide, YIYGSFK, was identified and has been proven to be an efficient and specific substrate for c-Src PTK. A relatively specific pseudosubstrate-based peptide inhibitor of c-Src PTK has been developed based on the structure of YIYGSFK. Furthermore, a fraction of the octapeptide library was screened for substrates of c-Abl PTK. Fourteen peptides have been identified. Four different consensus sequences have been obtained by comparing the amino acid sequences of these peptides, suggesting that c-Abl PTK may have a relatively broad substrate specificity. Using these identified peptides as substrates, intrinsic tyrosine kinase activities of c-Abl and Bcr-Abl PTKs were compared. The data have suggested that Bcr sequences may directly activate the kinase activity of Abl protein. In addition, roles of the SH2 and SH3 domains in the regulation of the intrinsic kinase activity of Bcr-Abl have been studied. The results have indicated that the SH2 domain rather than SH3 domain was required for the intrinsic kinase activity of Bcr-Abl oncoprotein. In summary, the use of the combinatorial peptide library method has revealed some new insights on the substrate specificity and recognition motifs of PTKs, and suggested that small linear peptide motifs are important in the substrate recognition and phosphorylation by PTKs although the other factors may also contribute to the substrate specificity of PTKs.
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42

Sidibeh, Cherno Omar. "Production and cleavage specificity determination of serine proteases mMCP-4, mMCP-5, rMCP-2 and two platypus serine proteases of the chymase locus." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-197088.

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Serine proteases are a family of enzymes with a wide array of functions across both eukaryotes and prokaryotes. Here we have attempted to produce the serine proteases rat mast cell protease 2 and mouse mast cell protease 5 in a culture of HEK 293 cells; and mouse mast cell protease 4, platypus granzyme B-like protease and platypus hypothetical protease in a baculovirus expression system. Following production we wanted to analyse these serine proteases using a phage display assay and a battery of chromogenic substrates.
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43

Risteli, M. (Maija). "Substrate specificity of lysyl hydroxylase isoforms and multifunctionality of lysyl hydroxylase 3." Doctoral thesis, University of Oulu, 2008. http://urn.fi/urn:isbn:9789514288296.

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Abstract Lysyl hydroxylase (LH) catalyzes the post-translational formation of hydroxylysines in collagens and collagenous proteins. Three lysyl hydroxylase isoforms, LH1, LH2 and LH3, have been identified from different species. In addition, LH2 has two alternatively spliced forms, LH2a and LH2b. The hydroxylysines have an important role in the formation of the intermolecular collagen crosslinks that stabilize the collagen fibrils. Some of the hydroxylysine residues are further glycosylated. In this thesis the substrate amino acid sequence specificities of the LH isoforms were analyzed using synthetic peptide substrates. The data did not indicate strict amino acid sequence specificity for the LH isoforms. However, there seemed to be a preference for some sequences to be bound and hydroxylated by a certain isoform. Galactosylhydroxylysyl glucosyltransferase (GGT) catalyzes the formation of glucosylgalactosylhydroxylysine. In this study, LH3 was shown to be a multifunctional enzyme, possessing LH and GGT activities. The DXD-like motif, characteristic of many glycosyltransferase families, and the conserved cysteine and leucine residues in the N-terminal part of the LH3 molecule were critical for the GGT activity, but not for the LH activity of the molecule. The GGT/LH3 protein level was found to be decreased in skin fibroblasts and in the culture media of cells collected from members of a Finnish epidermolysis bullosa simplex (EBS) family, which was earlier reported to have a deficiency of GGT activity. In this study, we showed that the reduction of enzyme activity is not due to a mutation or lower expression of the LH3 gene. Our data indicate that the decreased GGT/LH3 activity in cells has an effect on the deposition and organization of the key extracellular matrix components, collagen types VI and I and fibronectin, and these changes are transmitted to the cytoskeletal network. These findings underline LH3 as an important extracellular regulator.
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44

Tai, Guoying. "Structural determinants of CYP2C9's genetic variability, substrate specificity and dioxygen cleavage /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/8185.

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45

Kowlessur, Parikshant. "Engineering homoaromatic substrate specificity into aliphatic-specific Geobacillus pallidus RAPc8 nitrile hydratase." Thesis, University of the Western Cape, 2007. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_9829_1297833530.

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Geobacillus pallidus RAPc8 is a thermophilic nitrile-degrading isolate, obtained from thermal sediment samples of a New Zealand hot spring. The G. pallidus RAPc8 NHase gene has been cloned and expressed in E. coli. The recombinant NHase exhibits nitrile-degrading activity at 50 °
C, capable of degrading branched, linear and cyclic heteroaromatic nitrile substrates. However, no activity was found on homoaromatic nitrile substrates such as benzonitrile. In the present study, high levels of activity on benzonitrile were detected with a double mutant &beta
F52G&beta
F55L. Kinetic analysis on the mutant enzyme showed an 8-fold decrease in KM with benzonitrile (0.3mM) compared to acrylonitrile (2.6mM). Specificity constants (kcat/KM) of 5900 and 450 s-1.mM-1 were obtained for the double mutant on benzonitrile and acrylonitrile respectively. The amino acid residues lining the substrate channel were identified and the geometric dimensions measured. Cavity calculations revealed a 29% increase in volume and a 13% increase in inner surface area for the substrate channel of the double mutant when compared to the wild type. Surface representation of the wild type structure revealed two extended, curved channels, which are accessible to the bulk solvent from two locations in the heterodimer. The removal of the &beta
F52 may have contributed to the presence of a single channel with two opposing openings across the dimers with no internal blockage. Normal Mode Analysis calculations also indicate a higher intrinsic flexibility of the mutant relative tothe wild type enzyme. The increased flexibility within the mutant NHase could have introduced a functionally relevant aromatic substrate recognition conformation

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46

Knox, Stephen Richard. "Investigating the substrate specificity of foot and mouth disease virus 3C protease." Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445242.

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47

McKee, Lauren Sara. "Diversity in structure and substrate specificity of family 43 glycoside hydrolase enzymes." Thesis, University of Newcastle Upon Tyne, 2011. http://hdl.handle.net/10443/1239.

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There are strong drives to find a viable alternative to the use of petroleum as a transport fuel. Bioethanol presents an attractive option, but the long-term costs of producing this fuel from corn starch are now apparent. Second generation biofuels, derived from cellulosic plant cell walls, are a more acceptable alternative. A limiting factor in the economical utilisation of plant biomass is efficient saccharification of carbohydrates. The process is slowed by the chemical complexity of the substrate, notably the recalcitrant double substitution structures in arabinan and arabinoxylan. Reflecting this complex chemistry, microorganisms that degrade the wall synthesise an array of glycoside hydrolases. Several such organisms contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. To better understand the biological rationale behind the expansion in this family, the biochemical properties of the GH43 enzymes of a human gut symbiont, Bacteroides thetaiotaomicron, were investigated. Through cloning experiments, soluble protein was obtained for 25 enzymes. Activity screens uncovered several enzymes with a weak xylanase activity, three arabinoxylan-specific arabinofuranosidases, two endo-arabinanases and a novel arabinofuranosidase with specificity for α-1,2 side chains of singly and doubly substituted backbone residues. The crystal structure of a close homologue of the novel arabinofuranosidase is reported here. These data show how B. thetaiotaomicron deploys a combination of endo-acting and side chain-cleaving hydrolases to metabolise arabinan polysaccharides. Two GH43 enzymes (designated AXHd3s) have been found to target the double substitution structure in arabinoxylan. The crystal structure of the Humicola insolens AXHd3 was sought to understand this specificity, and is presented in complex with reaction products. Structural and mutagenic data were used to identify the mechanism by which the enzyme houses the O3-linked arabinofuranose in the active site, while exploiting the O2 appended arabinofuranose and asymmetrical xylan backbone as specificity determinants. Analysis of these data showed that orientation of the backbone, mediated by interactions with a conserved Tryptophan, positions the O3 arabinose into the active site. Modification of the rim of the active site pocket generated an AXHd3 variant that displayed both endo-xylanase and AXHd3 arabinofuranosidase activities. The introduction of additional catalytic functions into a biotechnologically relevant glycoside hydrolase provides a platform for evolving further, industrially significant, activities into the AXHd3 scaffold.
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48

Cotton, Thomas Richard. "An Investigation into the Sugar-substrate Specificity of the Sialic Acid Synthases." Thesis, University of Canterbury. Department of Chemistry, 2014. http://hdl.handle.net/10092/9920.

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The sialic acids represent a structurally and functionally diverse family of nine-carbon keto sugars. These compounds, derived structurally from neuraminic acid, are central to many of life’s processes at the molecular level. N-Acetylneuraminic acid (NeuNAc) is the most naturally abundant of the sialic acids and acts as the terminal residue of mammalian cell surface glycoconjugates. The negative charge and exposed location of NeuNAc makes this compound intrinsically linked to cellular signalling, recognition and adhesion events. While NeuNAc expression is typically restricted to eukaryotic phyla, some pathogenic bacteria have evolved to express NeuNAc on their own cell surfaces, allowing them to mimic the surface physiology of their mammalian host’s cells and evade detection by the immune system. In bacteria, NeuNAc biosynthesis proceeds via the aldol-like condensation reaction of phosphoenolpyruvate (PEP) with the sugar-substrate N-acetylmannosamine (ManNAc). Alternatively, in mammalian systems ManNAc is first phosphorylated to ManNAc 6-phosphate (ManNAc 6-P) before undergoing condensation with PEP to give NeuNAc 9-phosphate (NeuNAc 9-P). These PEP condensation reactions are catalysed by an evolutionarily related family of homo-dimeric (βα)₈ barrel enzymes (NeuNAc synthase in bacteria and NeuNAc 9-P synthase in mammals), referred to collectively as the sialic acid synthases. In addition to NeuNAc, several pathogenic bacteria synthesise a number of unique ‘bacterial sialic acids’, including legionaminic and pseudaminic acid, which are known to be essential for the motility and pathogenicity of some species. These alternate sialic acids are again biosynthesised via condensation reactions of PEP with variable sugar substrates, catalysed by additional members of the sialic acid synthase family. In chapter two of this thesis, I report a structural and functional characterisation of human NeuNAc 9-P synthase. Modelling and mutagenesis were used to delineate possible sugar-substrate binding modes, with a number of potentially important phosphate binding residues identified. Biophysical analysis reveals the human enzyme adopts a domain-swapped homo-dimeric conformation in solution, as previously observed for the analogous enzyme from Neisseria meningitidis. Chapter three details an investigation into the variable sugar-substrate specificity of mammalian and bacterial sialic acid synthases, which are apparently selective for their respective sugar-substrates depending entirely upon the presence or absence of the C-6 phosphate group. Bioinformatic analysis of bacterial and mammalian sequences revealed the β₂α₂ loop of the catalytic barrel as a putative sugar-substrate selectivity element. Substitution of the bacterial loop with the mammalian loop sequence however was alone insufficient to confer novel activity with the mammalian sugar-substrate. In chapter four, the β₂α₂ loop of the bacterial sialic acid synthases; legionaminic acid synthase (LegS) and pseudaminic acid synthase (PseS), was again identified as a structural element potentially involved in the sugar-substrate selectivity of these enzymes. Generation of chimeric LegS and PseS variants incorporating the β₂α₂ loop from bacterial NeuNAc synthase was unsuccessful at conferring novel NeuNAc synthase activity due to unforeseen disruption of PEP binding and quaternary structure. The work presented in this thesis provides a starting point from which to pursue a comprehensive understanding of the molecular basis of sugar-substrate specificity of the sialic acid synthases. An appreciation of how these enzymes achieve precise sugar-substrate specificity may provide the basis for their exploitation as therapeutic or biosynthetic targets.
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49

Fesser, Stephanie Marion. "Contribution of RNA binding proteins to substrate specificity in small RNA biogenesis." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-173105.

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50

Wang, Xing-Guo. "Alteration of substrate specificity in clostridial glutamate dehydrogenase by site-directed mutagenesis." Thesis, University of Sheffield, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387762.

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