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dc.contributor.authorLewis, Brett Eric
dc.date.accessioned2018-07-12T17:32:52Z
dc.date.available2018-07-12T17:32:52Z
dc.date.issued2004
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 64-09, Section: B, page: 4337.;Advisors: Vern L. Schramm.
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:3106727
dc.identifier.urihttps://hdl.handle.net/20.500.12202/663
dc.description.abstractIn this collection of manuscripts, we use isotope effects to explore the interaction between glucose and human brain hexokinase as a model for ligand or substrate binding. Whereas such binding isotope effects have been measured before, we conduct a full-molecule study using multiple singly-tritiated glucose compounds, finding effects at every backbone hydrogen atom (1.027 +/- 0.002, 0.927 +/- 0.0003, 1.027 +/- 0.004, 1.051 +/- 0.001, 0.988 +/- 0.001, and 1.065 +/- 0.003 for [1-t]-, [2-t]-, [3-t]-, [4-t]-, [5-t]-, and [6,6-t2]glucose, respectively). We utilize an extensive computational approach to understand these effects on the basis of known crystallographic data, and we find that for glucose binding to the hexokinase active site, isotope effects reflect partial deprotonation of the OH1, OH3, and OH4 hydroxyl groups by three active site carboxylates, direct steric impingement upon the backbone CH2 bond by Ser603, and at OH6, activation through partial deprotonation by Asp657 and assistance in positioning by Lys621.;We find that three of these isotope effects change upon addition of the ATP analog beta,gamma-CH2-ATP at 30 mM (15 Ki). The altered effects are 1.012, 0.998, and 1.032 for [1-t], [5-t], and [6,6-t 2], respectively. We attribute the alterations at H6 to partial satisfaction of O6 nucleophilicity by proximity to the gamma-phosphate group of ATP. This evidence of ground-state destabilization contributes to our present picture of hexokinase function.;Proving the existence of these effects requires measurement of isotopic effects on the anomeric equilibrium constant between glucose forms in water. We measure these deuterium equilibrium isotope effects using 13C-NMR spectroscopy and find these effects: 1.043 +/- 0.004, 1.027 +/- 0.005, 1.027 +/- 0.004, 1.001 +/- 0.003, 1.036 +/- 0.004, and 0.998 +/- 0.004 on the equilibrium constant, Kbeta/alpha, in [1-d], [2-d], [3-d], [4-d], [5-d], and [6,6-d2]-labelled sugars, respectively. Extensive computational modelling explains the effects by invoking the anomeric effect at H1, different hydroxyl torsional angle preferences at H2/OH2, and intramolecular steric interaction between the axial O1 in alpha-glucose and the H3 and H5 atoms themselves. (Abstract shortened by UMI.).
dc.publisherProQuest Dissertations & Theses
dc.subjectBiochemistry.
dc.titleGlucose in water at neutral and high pH and bound to human brain hexokinase via isotope effects and computational modeling
dc.typeDissertation


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