Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
The chemical and spectroscopic properties of 6-fluoropyridoxal 5'-phosphate, of its Schiff base with valine and of 6-fluoropyridoxamine 5'-phosphate have been investigated. The modified coenzymes have also been combined with the apo form of cytosolic aspartate aminotransferase and the properties of the resulting enzymes and of their complexes with substrates and inhibitors have been recorded. Although the presence of the 6-fluoro substituent reduces the basicity of the ring nitrogen over 10,000-fold, the modified coenzymes bind predominately in their dipolar ionic forms as do the natural coenzymes. Enzyme containing the modified coenzymes binds substrates and dicarboxylate substrates normally, and has about 42% of the catalytic activity of the native enzyme. Fluorine-19 NMR measurements show that the ring nitrogen of bound 6-fluoropyridoxamine phosphate is protonated at pH 7 or below but becomes deprotonated at high pH around a pKa of 8.2. The bound 6-fluoropyridoxal phosphate, which exists as a Schiff base with a dipolar ionic ring at high pH beomes protonated with a pKa of 7.1, corresponding to the pKa of 6.4 in the native enzyme. Below this pKa a single ('19)F resonance is seen corresponding to keoenamine and enolimine tautomers of the Schiff base. The tautomeric ratio and chemical shift is altered upon binding of dicarboxylate inhibitors indicating that during the rapid tautomerization a proton is synchronously moved from the ring nitrogen (in the ketoenamine tautomer) onto the aspartate 222 carboxylate (in the enolimine tautomer). Titration of the ('19)F NMR resonance with substrates and inhibitors can be related to exchange rates and equilibrium constants;A fluorescent derivative of lysine 258 isolated from the active site of aspartate aminotransferase modified by treatment of the apoenzyme with pyridoxal 5'-sulfate has been characterized as a substituted 2H-pyrrolo 3,4-c pyridine. Similar pyrrolopyridines are produced in up to 20% yield by reaction of pyridoxal sulfate with simple alkylamines or with amino acids. The reaction with lysine forms two products, one of which is identical to the isolated chromophore. The pyrrolopyridine derived from ethylamine has been characterized by ('1)H and ('13)C NMR, UV-visible, and mass spectroscopy.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Robert D. Scott
Scott, Robert D., "6-fluoropyridoxal phosphate and pyridoxal sulfate in aspartate aminotransferase " (1985). Retrospective Theses and Dissertations. 7885.