Isotopic investigations of redox enzyme mechanisms
Abstract
Lipoamide dehydrogenase is a flavoprotein which catalyses the reversible oxidation of dihydrolipoamide. The rate-limiting nature of proton transfer steps in both the forward and reverse reactions catalyzed by the pig heart enzyme was investigated using a combination of alternate substrates and solvent kinetic isotope effect (SKIE) studies. Plots of kinetic parameters against mole fraction D{dollar}\sb2{dollar}O yielded linear proton inventories in all cases. SKIE measurements performed in the reverse direction using NADH as the variable substrate showed normal SKIE's on V and {dollar}V/K\sb{lcub}\rm NADH{rcub}{dollar} when lipoamide, lipoic acid, or oxidized DTT were present at fixed saturating concentrations. With the disulfides as the variable substrates, normal SKIE's on V/K were also observed. Using DTNB as the variable substrate, an inverse SKIE was observed on V/K with no SKIE on V. The solvent equilibrium isotope effect was determined to be 0.438, which allowed for the calculation of the fractionation factor for the thiol moieties of dihydrolipoamide of 0.526.;Trypanothione reductase catalyzes the reduction of trypanothione (T(S){dollar}\sb2).{dollar} The pH dependence of the kinetic parameters V and V/K has been determined for the T. congolense enzyme. Both {dollar}V/K\sb{lcub}\rm NADH{rcub}{dollar} and V decreased as single groups exhibiting pK values of 8.87 {dollar}\pm{dollar} 0.09 and 9.45 {dollar}\pm{dollar} 0.07, respectively, were deprotonated. {dollar}V/K\sb{lcub}\rm T(S)2{rcub}{dollar} decreased as two groups exhibiting experimentally indistinguishable pK values of 8.74 {dollar}\pm{dollar} 0.03 were deprotonated. Variable magnitudes of the primary deuterium kinetic isotope effects (PDKIE's) on pyridine nucleotide oxidation were observed on V and V/K when different pyridine nucleotides substrates were used. A SKIE was observed on V only when cNADPH was the variable substrate, and a plot of this parameter against mole fraction D{dollar}\sb2{dollar}O was linear and yielded a value of 1.27 {dollar}\pm{dollar} 0.01 for {dollar}\sp{lcub}\rm D20{rcub}V.{dollar}.;Glycerol dehydrogenase from Cellulomonas catalyzes the reversible oxidation of glycerol to form dihydroxyacetone. Initial velocity, product, and dead end inhibition studies performed at pH 8.8 and 7.0 for the forward and reverse reactions, respectively, supported on ordered kinetic mechanism with NAD{dollar}\sp+{dollar} binding first and NADH released last. (Abstract shortened with permission of author.).
Permanent Link(s)
https://ezproxy.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:9219055https://hdl.handle.net/20.500.12202/3422
Citation
Source: Dissertation Abstracts International, Volume: 53-05, Section: B, page: 2295.;Advisors: John S. Blanchard.