Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.590328
Title: Dihydrofolate reductase and the physical basis of enzyme catalysis
Author: Behiry, Enas Mamdouh
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2013
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Abstract:
Dihydrofolate reductase (DHFR) is the enzyme that catalyses the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) in the presence of the cofactor reduced nicotinamide adenine dinucleotide phosphate (NADPH). The DHFR catalysed reaction has often been used to study enzymatic tunnelling and the contribution of protein dynamics to catalysis. To gain a better understanding of such phenomena and to investigate the key elements of structural adaptation in DHFR, in this thesis the hydride transfer reaction of DHFR from Moritella profunda (MpDHFR), a cold adapted enzyme, was studied and compared to the mesophilic and extensively studied enzyme from Escherichia coli (EcDHFR) and the thermophilic enzyme from Thermotoga maritima (TmDHFR). Chapter 1 gives a brief introduction to the thesis. Description of the materials and methods used in evaluating this work is reported in Chapter 2. In Chapter 3, the steady state and pre-steady state temperature dependences of the kinetic isotope effect (KIE) for the MpDHFR catalysed reaction was elevated, compared to data obtained for the mesophilic and the thermophilic DHFR homologues and the results interpreted according to the environmentally coupled tunnelling model. The work presented in Chapters 4 and 5 has investigated the role of dynamics during catalysis by DHFR using site directed mutagenesis. In Chapter 4, mutations were created in the GH loop for both EcDHFR and MpDHFR to elucidate the role of the occluded conformation during catalysis by DHFR. In Chapter 5, different MpDHFR and EcDHFR variants in the highly mobile M20 loop were generated and their temperature dependences of KIE were studied in addition to studying the two variants MpDHFR-G123V and MpDHFR-D124N in the catalytically important FG loop. The results obtained suggest that MpDHFR does not undergo the dynamical loop movements that have been recognized previously for EcDHFR in spite of following the same catalytic cycle. Further findings were found which contradict the current models that relate protein dynamics to catalysis efficiency, thus modifying these models has become essential. Chapter 6 has focused on studying the effect of different denaturants/salt concentrations on MpDHFR chemical step. Finally, a summary of the work presented in this thesis and future guidelines are provided in Chapter 7.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.590328  DOI: Not available
Keywords: QD Chemistry
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