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

dehaloperoxidase;heme enzyme;trihalophenol;Y39F;H56R;H90G;DHP;HRP

Chaudhary, Chelsea Erin ; danial feildheim, Committee Member,steven lommel, Committee Member,Stefan Franzen, Committee Chair,Chaudhary, Chelsea Erin ; danial feildheim ; Committee Member ; steven lommel ; Committee Member ; Stefan Franzen ; Committee Chair

University:North Carolina State University

关键词: dehaloperoxidase;    heme enzyme;    trihalophenol;    Y39F;    H56R;    H90G;    DHP;    HRP;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/351/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

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.