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dc.contributor.authorWong, Kenny K.
dc.date.accessioned2018-07-12T18:31:36Z
dc.date.available2018-07-12T18:31:36Z
dc.date.issued1990
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 51-02, Section: B, page: 7300.;Advisors: John S. Blanchard.
dc.identifier.urihttps://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:9017591
dc.identifier.urihttps://hdl.handle.net/20.500.12202/3305
dc.description.abstractGlutathione reductase catalyzes the pyridine nucleotide dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). The kinetic mechanism is ping-pong with the enzyme undergoing reduction in the reductive half-reaction and oxidation in the oxidative half-reaction. The enzyme is important in maintaining high levels of GSH in the cell. Solvent kinetic isotope effect (SKIE) studies on the human erythrocyte glutathione reductase have revealed a rate-limiting proton transfer step in the overall reaction which exhibits a solvent kinetic isotope effects of 2.5 on V{dollar}\sb{lcub}\rm max{rcub}{dollar} only. The plots of the V{dollar}\sb{lcub}\rm max{rcub}{dollar} versus mole fraction D{dollar}\sb2{dollar}O were linear. When an asymmetric disulfide analog of GSSG, containing a resonance-stabilized thionitrobenzoate moiety (GSSNB) was used as substrate, identical SKIEs on both V{dollar}\sb{lcub}\rm max{rcub}{dollar} and V{dollar}\sb{lcub}\rm max{rcub}{dollar}/K{dollar}\sb{lcub}\rm m{rcub}{dollar} were observed, which were linear versus mole fraction D{dollar}\sb2{dollar}O. When DTNB (Ellman's reagent) was used as a substrate, SKIEs were unity. The linear plots of the kinetic parameters versus D{dollar}\sb2{dollar}O for NADPH, GSSG and GSSNB suggested that the rate-limiting step is a single proton transfer step. These data, along with the structural information based on crystallographic studies, allowed us to conclude that the rate-limiting proton transfer step lies in the oxidative-half reaction. This step is between the first leaving glutathione thiolate and His-467{dollar}\sp\prime{dollar}:Glu-472{dollar}\sp\prime{dollar} ion pair.;Hydride transfer in the reductive half-reaction was examined using steady-state and pre-steady-state primary deuterium kinetic isotope effects, measured with NADH and (4S)-(4-{dollar}\sp2{dollar}H) NADH as substrates for the spinach, yeast, E. coli, and human erythrocyte glutathione reductases. These studies revealed differences in the magnitudes of the intrinsic primary deuterium kinetic isotope effect on hydride transfer. Corresponding primary tritium KIEs argue that the isotope effect was fully expressed and that hydride transfer was completely rate-limiting when NADH was used as a substrate. These differences suggested that the transition-states for the four enzymes are different, even though they catalyze the same reaction between the same redox partners. We propose that these differences are due to differential stabilization of the transition state for hydride transfer by each enzyme.
dc.publisherProQuest Dissertations & Theses
dc.subjectBiochemistry.
dc.titleMechanistic analysis of glutathione-dependent enzymes
dc.typeDissertation


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