Nucleotide Inhibition of Glyoxalase II Metadata
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- Main Title Nucleotide Inhibition of Glyoxalase II
Author: Gillis, Glen SCreator Type: Personal
Chair: Norton, Scott J.Contributor Type: PersonalContributor Info: Major Professor
Committee Member: Massarachia, Ruth AnneContributor Type: Personal
Committee Member: Donahue, Manus J.Contributor Type: Personal
Committee Member: Chapman, Kent D.Contributor Type: Personal
Name: University of North TexasPlace of Publication: Denton, Texas
- Creation: 1999-05
- Content Description: The glyoxalase system mediates the conversion of methylglyoxal, a toxic ketoaldehyde, to D-lactic acid. The system is composed of two enzymes, glyoxalase I (Glo-I) and glyoxalase II (Glo-II), and exhibits an absolute requirement for a catalytic quantity of glutathione (GSH). Glo-I catalyzes the isomerization of a hemithioacetal, formed non-enzymatically from methylglyoxal and GSH, to the corresponding a -D-hydroxyacid thioester, s-D-lactoylglutathione (SLG). Glo-II catalyzes the irreversible breakdown of SLG to D-lactate and GSH. We have observed that ATP or GTP significantly inhibits the Glo-II activity of tissue homogenates from various sources. We have developed a rapid, one step chromatography procedure to purify Glo-II such that the purified enzyme remains "sensitive" to inhibition by ATP or GTP (Glo-II-s). Studies indicate that inhibition of Glo-II-s by nucleotides is restricted to ATP, GTP, ADP, and GDP, with ATP appearing most effective. Kinetics studies have shown that ATP acts as a partial non-competitive inhibitor of Glo-II-s activity, and further suggest that two kinetically distinguishable forms of the enzyme exist. The sensitivity of pure Glo-II-s to nucleotide inhibition is slowly lost on storage even at -80° C. This loss is accelerated at higher temperatures or in the presence of ATP. Kinetics studies on the resultant "insensitive" enzyme (Glo-II-i) show that a significant reduction of the affinity of the enzyme for the substrate, SLG, occurs and further suggest that only one form of the enzyme is kinetically distinguishable after "de-sensitization". Tryptophan fluorescence studies of the two enzyme preparations suggest that a subtle conformational change in the enzyme has occurred during de-sensitization. We have also observed that Glo-II-i is "resensitized" to nucleotide inhibition after incubation in the presence of a reagent that reduces disulfide bonds. The resensitized enzyme exhibits an increased KM value similar to that of the original Glo-II-s. Kinetics studies show that ATP or GTP again act as partial non-competitive inhibitors of the resensitized enzyme and suggest that only one form of the enzyme is present. The physiological significance of the two enzyme forms is discussed.
- Library of Congress Subject Headings: Glyoxalase.
- Library of Congress Subject Headings: Nucleotides.
- Library of Congress Subject Headings: Glutathione.
- Keyword: glyoxalase I
- Keyword: glutathione
- Keyword: ATP
- Keyword: GTP
- Keyword: glyoxalase II
Name: UNT Theses and DissertationsCode: UNTETD
Name: UNT LibrariesCode: UNT
- Rights Access: public
- Rights License: copyright
- Rights Holder: Gillis, Glen S
- Rights Statement: Copyright is held by the author, unless otherwise noted. All rights reserved.
- Thesis or Dissertation
- OCLC: 45003473
- UNT Catalog No.: b2226120
- Archival Resource Key: ark:/67531/metadc2183
- Degree Name: Doctor of Philosophy
- Degree Level: Doctoral
- Degree Discipline: Biochemistry
- Academic Department: Department of Biological Sciences
- Degree Grantor: University of North Texas