The use of carbon-13 nuclear magnetic resonance spectroscopy to elucidate enzyne structure and reactivity
The use of carbon-13 nuclear magnetic resonance spectroscopy to elucidate enzyne structure and reactivity
The major objective of this research has been to combine the techniquesof enzyme inhibition studies and 13C nuclear magnetic resonance spectroscopy in order to provide a method to investigate reactive functional groups, and the specific environment in which they occur, within an enzyme. This was performed by introduction of a covalent label into an enzyme by reaction with a 13C-enriched electrophile, and subsequent determination by 13C nuclear magnetic resonance spectroscopy.Initially, amino-acid model compounds and tripeptides were prepared and reacted with electrophilic inhibitors in order to correlate the 13C nuclear magnetic resonance chemical shift data obtained with that of the 13C-enriched resonance positions in the enzyme. In addition, the effect of solvent and pH on the 13C chemical shifts was investigated. Ribonuclease A has been reacted with 90%[2-13C]-bromoacetic acid. Examination of the 13C nuclear magnetic resonance spectra of the specifically enriched enzyme enabled the sites of reaction to be identified; these were at histidine to form mono- and di-carboxymethyl derivatives and at methionine to form the sulphonium salt. The time course and sequence of alkylation was monitored by performing the inhibition reaction in a sample tube in the probe of the 13C nuclear magnetic resonance spectrometer. Partially-relaxed Fourier transform 13C nuclear magnetic resonance spectroscopy has allowed a quantitative description of the motion of the 13C-enriched nuclei in native,and acid- and urea-denatured ribonuclease, thus providing information concerning the constraints on motion imposed by the tertiary structure of the enzyme. In addition, the effect of denaturation and renaturation on the 13C nuclear magnetic resonance spectra was observed. The study has also been extended to glyceraldehyde-3-phosphate dehydrogenase and 6-aminolevulinic acid dehydratase.
University of Southampton
1976
Climie, Ian James Guy
(1976)
The use of carbon-13 nuclear magnetic resonance spectroscopy to elucidate enzyne structure and reactivity.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The major objective of this research has been to combine the techniquesof enzyme inhibition studies and 13C nuclear magnetic resonance spectroscopy in order to provide a method to investigate reactive functional groups, and the specific environment in which they occur, within an enzyme. This was performed by introduction of a covalent label into an enzyme by reaction with a 13C-enriched electrophile, and subsequent determination by 13C nuclear magnetic resonance spectroscopy.Initially, amino-acid model compounds and tripeptides were prepared and reacted with electrophilic inhibitors in order to correlate the 13C nuclear magnetic resonance chemical shift data obtained with that of the 13C-enriched resonance positions in the enzyme. In addition, the effect of solvent and pH on the 13C chemical shifts was investigated. Ribonuclease A has been reacted with 90%[2-13C]-bromoacetic acid. Examination of the 13C nuclear magnetic resonance spectra of the specifically enriched enzyme enabled the sites of reaction to be identified; these were at histidine to form mono- and di-carboxymethyl derivatives and at methionine to form the sulphonium salt. The time course and sequence of alkylation was monitored by performing the inhibition reaction in a sample tube in the probe of the 13C nuclear magnetic resonance spectrometer. Partially-relaxed Fourier transform 13C nuclear magnetic resonance spectroscopy has allowed a quantitative description of the motion of the 13C-enriched nuclei in native,and acid- and urea-denatured ribonuclease, thus providing information concerning the constraints on motion imposed by the tertiary structure of the enzyme. In addition, the effect of denaturation and renaturation on the 13C nuclear magnetic resonance spectra was observed. The study has also been extended to glyceraldehyde-3-phosphate dehydrogenase and 6-aminolevulinic acid dehydratase.
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Published date: 1976
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Local EPrints ID: 462531
URI: http://eprints.soton.ac.uk/id/eprint/462531
PURE UUID: d261df6f-e1e8-43dd-82e4-fb5d5b4b2826
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Date deposited: 04 Jul 2022 19:16
Last modified: 04 Jul 2022 19:16
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Author:
Ian James Guy Climie
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