Mechanistic Studies of the Sheep Liver 6-Phosphogluconate Dehydrogenase and cDNA Cloning

dc.contributor.advisorNeeraj Agarwal
dc.contributor.committeeMemberRobert Easom
dc.contributor.committeeMemberStephen R. Grant
dc.creatorPrice, Nancy E.
dc.description.abstractPrice, Nancy E., Mechanistic Studies of the Sheep Liver 6-Phosphogluconate Dehydrogenase and cDNA Cloning. Doctor of Philosophy (Biomedical Sciences), July, 1996, 124 pp., 5 tables, 28 Figures, 2 appendices, bibliography, 45 titles. A kinetic characterization of sheep liver 6-phosphogluconate dehydrogenase including product and dead-end inhibition patterns, primary deuterium isotope effects, and the pH dependence of kinetic parameters has been completed in order to determine the kinetic mechanism, and chemical mechanism of the enzyme. A rapid equilibrium random kinetic mechanism has been proposed, with product and dead-end inhibition patterns both being symmetric. Primary deuterium isotope effects were equal on V and V/K, confirming a rapid equilibrium mechanism, and indicate that hydride transfer is at least partially rate limiting in the overall reaction. The maximum velocity is pH dependent, decreasing at low and high pH with slopes of 1 and -1, respectively. The V/KNADP and V/K6PG also decrease at low and high pH with slopes of 1 and -1. The pH rate profiles are consistent with a general acid/general base mechanism where the catalytic residues are involved in binding. Reverse protonation states between the general acid and the general base is proposed where an unprotonated general base accepts a proton from the C-3 hydroxyl of 6PG concomitant with hydride transfer followed by decarboxylation of the resulting 3-keto intermediate to give an enediol which is protonated by the general acid to form ribulose-5-phosphate. The pH dependence of the pKi profile of the inhibitory analog 5-phosphoribonate decreases at low and high pH with slopes of 1, and -1 respectively, and suggests that intrinsic pKs are observed in the V/K profiles. The pKs of both the general base and general acid in the E:6PG complex appears to be perturbed such that the general base pK decreases slightly, and the pK of the general acid increases slightly, as a result of direct interaction with 6PG. Additionally, in preparation for site-directed mutagenesis, cDNA clones for sheep liver 6PHDH were obtained by RT-PCR.
dc.subjectAnimal Sciences
dc.subjectAnimal Structures
dc.subjectCell and Developmental Biology
dc.subjectCell Biology
dc.subjectCellular and Molecular Physiology
dc.subjectComparative and Laboratory Animal Medicine
dc.subjectGenetic Phenomena
dc.subjectGenetic Processes
dc.subjectGenetics and Genomics
dc.subjectGenetic Structures
dc.subjectInvestigative Techniques
dc.subjectLife Sciences
dc.subjectMedical Cell Biology
dc.subjectMedical Genetics
dc.subjectMedical Sciences
dc.subjectMedicine and Health Sciences
dc.subjectMolecular Biology
dc.subjectOther Animal Sciences
dc.subjectOther Cell and Developmental Biology
dc.subjectOther Genetics and Genomics
dc.subjectOther Veterinary Medicine
dc.subjectSheep liver
dc.subjectmechanistic study
dc.subject6-phosphogluconate dehydrogenase
dc.subjectcDNA cloning
dc.subjectpH dependence
dc.titleMechanistic Studies of the Sheep Liver 6-Phosphogluconate Dehydrogenase and cDNA Cloning
dc.type.materialtext School of Biomedical Sciences Sciences of North Texas Health Science Center at Fort Worth of Philosophy


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