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Tuning the Substrate Specificity of the Glutathione Transferase GstB from Escherichia coli via Site-directed Mutagenesis

Moore, Jennifer Marie

Abstract Details

2017, Master of Science in Chemistry, Youngstown State University, Department of Chemistry.
Glutathione transferases (GSTs) are detoxication enzymes that are widely distributed in nature. They fulfill their protective roles by catalyzing the conjugation of the tripeptide glutathione to both endogenous and xenobiotic electrophiles. Eukaryotic GSTs are the subject of thorough investigation, while the prokaryotic enzymes remain relatively unexplored. GstB is a glutathione transferase from Escherichia coli that is known to detoxify bromoacetate, a water disinfection by-product. This work has served to expand the substrate scope for GstB. Site-directed mutagenesis of the electrophile binding site residue arginine 119 was performed, generating the alanine, glutamine, histidine, and serine enzyme variants. The activities of the mutants toward a range of electrophiles were evaluated to investigate the impact of the amino acid substitutions on substrate specificity. Initial activity screening results indicate that some mutants display rate enhancement for acrylate and iodoacetamide conjugation. Kinetic parameters with iodoacetamide suggest that certain mutants are more catalytically efficient and resistant to inhibition compared to the wild type enzyme. The finding that amino acid substitution at position 119 can modify GstB substrate specificity provides support for pollutant-targeted bioremediation strategies.
Nina Stourman, PhD (Advisor)
Michael Serra, PhD (Committee Member)
Peter Norris, PhD (Committee Member)
159 p.

Recommended Citations

Citations

  • Moore, J. M. (2017). Tuning the Substrate Specificity of the Glutathione Transferase GstB from Escherichia coli via Site-directed Mutagenesis [Master's thesis, Youngstown State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1502910733353098

    APA Style (7th edition)

  • Moore, Jennifer. Tuning the Substrate Specificity of the Glutathione Transferase GstB from Escherichia coli via Site-directed Mutagenesis . 2017. Youngstown State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ysu1502910733353098.

    MLA Style (8th edition)

  • Moore, Jennifer. "Tuning the Substrate Specificity of the Glutathione Transferase GstB from Escherichia coli via Site-directed Mutagenesis ." Master's thesis, Youngstown State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1502910733353098

    Chicago Manual of Style (17th edition)