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Title: Aspects of catalysts in trimethylamine dehydrogenase : substrate inhibition, H-tunnelling and substrate design
Author: Roberts, Peter
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2000
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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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available