Identifier

etd-0409103-143112

Degree

Master of Science (MS)

Department

Biological Sciences

Document Type

Thesis

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 van’t Hoff plot in H2O was biphasic showing an apparent transition temperature of 20°C, 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 25°C 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 25°C. 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.

Date

2003

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Grover Waldrop

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