Carbonic anhydrase : how does this zinc enzyme work? carbon-13 magnetization transfer studies of carbon dioxide-bicarbonate exchange / Doris Ohliger.
xi, 113 p. : ill. ; 29 cm.
- Medical subjects:
- Biochemistry and Molecular Biophysics.
- Local subjects:
- Penn dissertations -- Biochemistry and molecular biophysics.
Biochemistry and molecular biophysics -- Penn dissertations.
- A 13C NMR saturation transfer technique was used to measure equilibrium reaction rates for the hydration of 13CO2 and the dehydration of H13CO3- as catalysed by carbonic anhydrase II. The purpose of these studies was to validate the 13C saturation transfer technique for future in vitro and in vivo studies of the reversible hydration and dehydration reactions. Initial studies were undertaken with bovine carbonic anhydrase II (CA II), a zinc enzyme. Conditions varied were CA II or catalyst concentration, the concentration of bicarbonate, the pH, the temperature, and the solvent. Equilibrium reaction rates obtained were determined to be 8.5 x 10 5 seconds-1 for the hydration reaction and 2.6 x 105 seconds-1 for the dehydration reaction and these are consistent with previous reports. The advantage of using this kinetic method is that first order reaction rates are measured at chemical equilibrium and that both the forward and reverse reactions are measured independently. The kinetic studies of CA II presented in this thesis, combined with observations of the X-ray protein structure, suggest that a customized alignment of water molecules is found within the active site cavity of each distinct CA isozyme that contributes to the variability in reaction rate among the distinct CA isozymes. These analyses demonstrate a novel use of the 13C saturation transfer technique that is applicable to both in vitro and in vivo studies of CA isozymes. Mechanistic information obtained from in vitro studies can be combined with in vivo observations of bicarbonate-carbon dioxide exchange in skeletal muscle as catalysed by CA III and in the mitochondrion as catalysed by CA V to further delineate their metabolic functions.
- Supervisor: Robert E. Forster.
Thesis (Ph.D. in Biochemistry and Molecular Biophysics) -- University of Pennsylvania, 2001.
Includes bibliographical references.
- Local notes:
- University Microfilms order no.: 3015351.
- Forster, Robert E., advisor.
University of Pennsylvania.
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