Title page for ETD etd-0409103-143112


Type of Document Master's Thesis
Author Lord, Brett K.
Author's Email Address blord1@lsu.edu
URN etd-0409103-143112
Title Temperature Dependent Kinetics of Biotin Carboxylase
Degree Master of Science (M.S.)
Department Biochemistry (Biological Sciences)
Advisory Committee
Advisor Name Title
Grover Waldrop Committee Chair
Anne Grove Committee Member
Sue Bartlett Committee Member
Keywords
  • temperature dependence
  • biotin carboxylase
Date of Defense 2003-04-08
Availability unrestricted
Abstract
Acetyl-CoA carboxylase catalyzes the first committed step in long chain fatty acid biosynthesis. In Escherichia coli, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxytransferase. The biotin carboxylase component has served for many years as a model for mechanistic studies devoted toward understanding biotin-dependent carboxylases. Studies of the temperature dependence, temperature dependence of the kinetic solvent isotope effect and thermodynamics of biotin carboxylase are reported. Analysis of the vant Hoff plot in H2O was biphasic showing an apparent transition temperature of 20C, with corresponding DH values of 4.55 1.84 kcal/mol below the transition temperature and 1.59 0.16 kcal/mol above the transition temperature, respectively, suggesting a conformational change is occurring at this temperature. Biphasic Arrhenius and Eyring plots in D2O showed an apparent transition temperature at 25C with corresponding Ea and DH values of 16.35 0.90 kcal/mol and 15.86 0.85 kcal/mol below the transition temperature, respectively, and Ea and DH values of 4.01 1.15 kcal/mol and 3.37 1.06 kcal/mol above the transition temperature, respectively. This break in the plots is suggestive of either a conformational change or a change in the rate-determining step occurring at 25C. Kinetic solvent isotope effects were used to distinguish between these two possibilities. The results of the kinetic solvent isotope effect suggest a change in the rate-determining step as a function of temperature is occurring and is not due to a conformational change. Analysis of Arrhenius preexponential factors (AH/AD) determined from the temperature dependence of the kinetic solvent isotope suggests both hydrogen and deuterium tunneling in biotin carboxylase.
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