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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 pKa 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.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Raftery, Michael A.
Thesis Committee:
  • Raftery, Michael Augustine
Defense Date:14 December 1972
Additional Information:The thesis title listed in 1973 commencement program -- I. ³¹p and ¹³c NMR Studies of NAD and Related Compounds. II. ¹⁹f NMR Studies of Rabbit Muscle Glyceraldehyde-3-Phosphate Dehydrogenase Covalently Labeled with a Trifluoromethyl Group -- varies from the actual thesis title.
Record Number:CaltechTHESIS:11122019-135233564
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:11122019-135233564
DOI:10.7907/0XYM-Z897
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:13570
Collection:CaltechTHESIS
Deposited By: Melissa Ray
Deposited On:14 Nov 2019 01:25
Last Modified:18 Dec 2020 19:33

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