Point Mutagenesis and Spectrosopic Probing of Dehaloperoxidase: Characterizing the Mechanism and Activity of the Heme Active Site of the Native Protein

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dc.contributor.advisor danial feildheim, Committee Member en_US
dc.contributor.advisor steven lommel, Committee Member en_US
dc.contributor.advisor Stefan Franzen, Committee Chair en_US
dc.contributor.author Chaudhary, Chelsea Erin en_US
dc.date.accessioned 2010-04-02T17:54:51Z
dc.date.available 2010-04-02T17:54:51Z
dc.date.issued 2003-07-25 en_US
dc.identifier.other etd-05162003-170737 en_US
dc.identifier.uri http://www.lib.ncsu.edu/resolver/1840.16/351
dc.description.abstract The research presented in this thesis focuses on DHP, the three mutants of DHP (Y39F, H56R, and H90G), and their relationship to well known enzymes such as HRP and myoglobin. Two methods of doing point mutations were performed on three DHP amino acids, which were near the active site and/or the heme, to understand more about the substrate binding activity and product formation of the native enzyme. Mutagenesis techniques employing the polymerase chain reaction (PCR), ligation, transformation, inoculation, and protein purification were carried out. Specifically, Y39F was developed due to its proximity to the substrate binding site and its hydrogen bond to the bound substrate. H56R and H90G are commonly studied mutants of myoglobin, which have been shown to decrease activity due to the changes in the R groups of the mutated amino acids. Activity assays for understanding the reaction of the heme in DHP were developed and performed on the native enzyme as well as Y39F, H56R, and H90G. The assays included using tri-halogenated phenols, hydrogen peroxide, and differing concentrations of enzyme in a pH 7 phosphate buffer. Spectroscopic probing on a multi wavelength ultraviolet/visible spectrometer revealed that all mutants and the native protein differ in the rate and amount of product formed as well as heme degradation (due to the peroxide). Singular Value Decomposition, SVD, calculations were carried out to single out the three components of the reaction (product growth, substrate consumption, heme shift and concentration change) and then the matrices were rotated by specified angles for further analysis. en_US
dc.rights I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. en_US
dc.subject dehaloperoxidase en_US
dc.subject heme enzyme en_US
dc.subject trihalophenol en_US
dc.subject Y39F en_US
dc.subject H56R en_US
dc.subject H90G en_US
dc.subject DHP en_US
dc.subject HRP en_US
dc.title Point Mutagenesis and Spectrosopic Probing of Dehaloperoxidase: Characterizing the Mechanism and Activity of the Heme Active Site of the Native Protein en_US
dc.degree.name MS en_US
dc.degree.level thesis en_US
dc.degree.discipline Chemistry en_US


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