I. ³¹P and ¹³C Nuclear Magnetic Resonance Studies of Nicotinamide Adenine Denucleotide and Related Compounds. II. ¹⁹F Nuclear Magnetic Resonance Studies of Rabbit Muscle Glyceraldehyde-3-Phosphate Dehydrogenase Covalently Labeled with a Trifluoromethyl Group

Citation

Blumenstein, Michael (1973) I. ³¹P and ¹³C Nuclear Magnetic Resonance Studies of Nicotinamide Adenine Denucleotide and Related Compounds. II. ¹⁹F Nuclear Magnetic Resonance Studies of Rabbit Muscle Glyceraldehyde-3-Phosphate Dehydrogenase Covalently Labeled with a Trifluoromethyl Group. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/0XYM-Z897. https://resolver.caltech.edu/CaltechTHESIS:11122019-135233564

Abstract

The ³¹P spectra of the reduced and oxidized forms of nicotinamide adenine dinucleotides as well as several related mono- and dinucleotides have been obtained and analyzed. From the spectral differences between the reduced and oxidized nucleotides, as well as from the determination of the pK_a values of the phosphate group in the mononucleotides, it is postulated that in the oxidized nucleotides there is an electrostatic interaction between the positively charged nitrogen of the pyridine ring and a negatively charged oxygen of the diphosphate backbone.

The natural abundance ¹³C spectra of nicotinamide adenine dinucleotide and related compounds have been recorded and assigned. These spectra yielded further evidence for the nicotinamide-phosphate interaction in oxidized nucleotides. Small effects on the ¹³C spectra were observed upon changes in pH or nucleotide concentration.

Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase has been reacted with bromotrifluoroacetone, leading to a modified protein containing a trifluoromethyl group at each of its four active site cysteines. Addition of NAD⁺ or NADH caused changes in the ¹⁹F spectrum arising from the bound trifluoromethyl group. Also changes in pH affected the ¹⁹F spectrum. At high pH, two resonances were observed, leading to the conclusion that the enzyme, which is composed of four subunits having identical amino acid sequences, exists as an α₂α₂' protein. The chemical shift changes observed in this study (up to 19 parts per million) are considerably larger than those previously observed in ¹⁹F nuclear magnetic resonance studies of proteins.

Measurements of the spin-spin and spin-lattice relaxation times of the enzyme bound trifluoromethyl group have been performed. It has been shown that the bound label has very little mobility, and interacts with solvent protons, as well as protons on other residues of the protein.

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