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|Title:||Aspects of catalysts in trimethylamine dehydrogenase : substrate inhibition, H-tunnelling and substrate design|
|Presented at:||University of Leicester|
|Abstract:||Trimethylamine dehydrogenase (TMADH) is an iron-sulphur flavoprotein that catalyses the demethylation of trimethylamine (TMA) to dimethylamine and formaldehyde. In this thesis, three aspects of TMADH enzymology have been addressed: the effect of substrate on the redox state of the enzyme, the quantum mechanical tunnelling of hydrogen during substrate C-H bond cleavage, and an attempt to improve specificity for the secondary amine DMA by rational protein engineering.;Substrate inhibition in TMADH has been studied using the native enzyme and active site mutants that show different degrees of substrate inhibition. Use of Fc+ and a photodiode array to directly observe the redox state of the enzyme during steady-state turnover has demonstrated that TMADH participates in two different redox cycles. At low TMA concentrations, the enzyme undergoes a 0/2 cycle, in which enzyme bound flavin is reduced by a maximum of two reducing equivalents; when inhibited at high TMA concentrations the enzyme undergoes a 1.3 cycle, in which enzyme bound flavin is reduced by either one or three reducing equivalents. Increasing the concentration of the electron acceptor Fc+ decreases the extent of inhibition. Excess substrate binding at the active site of 1-electron reduced enzyme stabilisers the semiquinone form of enzyme bound 6-S-cysteinyl FMN, preventing further reduction by substrate, causing excess substrate inhibition.;C-H bond cleavage in TMADH has been investigated with regard to determining the importance of H-tunnelling mediated by protein dynamics during catalysis. The results of this preliminary study are highly suggestive of H-tunnelling during catalysis by TMADH.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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