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Mutagenic Studies of Substrate Specificity and Stability of Paraoxonase-1

Harsch, Christina I K
http://rave.ohiolink.edu/etdc/view?acc_num=osu1325197473

Abstract Details

2011, Doctor of Philosophy, Ohio State University, Chemistry.
There are limited efficient methods in treating organophosphorus (OP)-poisoning through exposure to pesticides and nerve agents. Human paraoxonase-1 (PON1) is a calcium-dependent enzyme that is produced in the liver and is secreted into serum where it associates with HDL particles. It was discovered that PON1 is able to hydrolyze organophosphorus pesticides and nerve agents. In order to use PON1 in a potential bioscavenger therapeutic strategy, there are challenges that need to be addressed. Large-scale production of soluble protein and the improvement of catalytic activity are two of the most important challenges for PON1. Human PON1 is very unstable and not very soluble, most likely due to its physiological association with HDL particles. To address these concerns, recombinant chimeric forms of PON1 (G2E6 and G3C9) have been identified. G2E6 and G3C9 are chimeric forms of PON1, produced from directed evolution techniques using DNA shuffling methods with PON1 sequences from rabbit, mouse, rat and human. Both variants were identified through screening methods to discover active enzymes against substrates, including OP compounds. These enzymes are both more soluble and stable than human PON1. The sequences between human PON1 and the chimeric forms of PON1 are largely different in the surface residues, leaving the putative active sites the same for all three enzymes. In order to validate that G2E6 has similar activities to human PON1, we analyzed the aryl esterase and paraoxonase activities for both enzymes. They were also tested for VX and VR activity. Mutations were made within the active site as well as near the surface to determine differences in activities. It was concluded that the H115W mutant in G2E6 retained paraoxonase activity but increased with the human PON1. The aryl esterase activity was eliminated with the H115W in human PON1 but diminished in the G2E6 scaffold. The H115W mutation in human PON1 and in G2E6 showed an increase in activity against VX, with the G2E6 scaffold having only modest activities against nerve agents. These experiments demonstrated that G2E6 and human PON1 have different substrate specificities, despite their similarities in sequence. To examine this result more thoroughly, the H115 position in G2E6 was mutated with various representative amino acids (hydrophobic, polar, aromatic, charged). These mutants were tested for substrate specificity against various OP substrates (paraoxon, CMP, EMP, VX, and VR) as well as catalytic efficiency. We concluded that substrate specificity in regards to position 115 is dependent upon the residue present. The differences in activities further demonstrated the substrate specificity differences between human PON1 and G2E6. Of the 59 amino acid residue differences between G2E6 and human PON1, most of the mutations are on the surface. The exceptions are positions 192 and 166, which point into the active site. The positions were examined by mutagenic methods to study the catalytic efficiencies against substrates as well as the overall stability of the mutant enzymes. In examining the multi-mutant enzymes identified from collaborators in the Weizmann Institute, single point mutations were made in G3C9 to examine the importance of each of the individual mutation’s contributions to substrate specificity.
Thomas Magliery, PhD (Advisor)
Christopher Hadad, PhD (Committee Member)
Christopher Jaroniec, PhD (Committee Member)
Biochemistry; Chemistry
paraoxonase-1; PON1

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Citations

  • Harsch, C. I. K. (2011). Mutagenic Studies of Substrate Specificity and Stability of Paraoxonase-1 [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1325197473

    APA Style (7th edition)

  • Harsch, Christina. Mutagenic Studies of Substrate Specificity and Stability of Paraoxonase-1. 2011. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1325197473.

    MLA Style (8th edition)

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

    Chicago Manual of Style (17th edition)

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© 2011, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.