Solid-State 17O NMR as a New Probe to Study Acyl-Enzyme Intermediates
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Authors
Tang, Aaron W.
Date
2016-04-30
Type
thesis
Language
eng
Keyword
solid-state , Chymotrypsin , acyl-enzyme , magic-angle spinning , NMR , computation , 17O
Alternative Title
Abstract
This thesis reports on a combined solid-state 17O NMR and quantum mechanical study of three acyl-enzyme intermediates: p-N,N-dimethylaminobenzoyl-chymotrypsin (DAB-CHT), trans¬¬-¬o-methoxycinnamoyl-chymotrypsin (oMC-CHT), and trans-p-methoxycinnamoyl-chymotrypsin (pMC-CHT). At pH 7.8, the three acyl-enzyme intermediates were found to undergo hydrolysis (deacylation) with the following rate constants: DAB-CHT, 6.4 ± 0.1 × 10-6; oMC-CHT, 2.8 ± 0.1 × 10-4 s-1; pMC-CHT, 8.5 ± 0.3 × 10-3 s-1. Analysis of solid-state 17O NMR spectra obtained under the magic angle spinning (MAS) condition at 21 T for the three acyl-enzyme intermediates yields the following isotropic chemical shift (δiso), quadrupolar coupling constant (CQ), and asymmetry parameter (ηQ): DAB-CHT, δiso = 316 ± 2 ppm, CQ = 10.0 ± 1 MHz, ηQ = 0.6 ± 0.2; oMC-CHT, δiso = 316 ± 2 ppm, CQ = 9.2 ± 1 MHz, ηQ = 0.6 ± 0.2; and pMC-CHT, δiso = 316 ± 2, CQ = 7.0 ± 0.5 MHz, ηQ = 0.8 ± 0.2. With the aid of quantum mechanical calculations, these 17O NMR parameters were interpreted as to reflect the variations in hydrogen bonding interactions between the carbonyl (C=O) functional group of the acyl moiety and the two NH groups from the protein backbone in the so-called oxyanion hole. This work represents the first use of solid-state 17O NMR spectroscopy to study acyl-enzyme intermediates and demonstrates the great potential of this new technique.
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Thesis (Master, Chemistry) -- Queen's University, 2016-04-29 19:42:38.179
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ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
Creative Commons - Attribution - CC BY
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.