Use this URL to cite or link to this record in EThOS:
Title: Probing the basis of catalysis of dihydrofolate reductase
Author: Adesina, Aduragbemi
ISNI:       0000 0004 7962 1263
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2018
Availability of Full Text:
Access from EThOS:
Access from Institution:
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 the contribution of protein dynamics to enzyme catalysis. To better understand protein dynamics and to investigate how it relates to electrostatic changes in DHFRs, the dynamics of DHFR from human (HsDHFR), Escherichia coli (EcDHFR), the cold-adapted Moritella profunda (MpDHFR), the moderately thermophilic Geobacillus stearothermophilus (BsDHFR) and the thermophilic enzyme from Thermatoga maritima (TmDHFR) are studied using kinetic isotope effects and vibrational Stark effects spectroscopy. Chapter 1 gives a brief introduction to the thesis. In Chapter 2, the purification of HsDHFR was optimised due to the kinetic complexity introduced by the purification strategy. The study showed that using a truncated ligand lessens the complexity that has prevented detailed kinetic characterisation of HsDHFR for three decades. The work presented in Chapter 3 involves optimisation of kinetic conditions to reveal the chemical step of HsDHFR and to study the effects of dynamics on it. The result showed that tunnelling did not contribute to the dynamics of HsDHFR, in contrast to reports for its bacterial homologs. It also showed that protein dynamics are not coupled to the chemical step of the enzyme at its physiological temperature. In Chapter 4, vibrational Stark effect spectroscopy was employed to study ligand interaction in EcDHFR, its conformationally impaired variant EcDHFR-S148P, BsDHFR, MpDHFR and TmDHFR. A site-specific nitrile probe was inserted at a conserved position to monitor electrostatic changes in the enzymes. The results obtained suggest that electrostatics in DHFRs are generally conserved despite exhibiting different dynamic flexibilities. In addition, the preorganisation of cofactor-substrate interaction in the enzyme was found to play a major role in their catalysis. A summary of the work presented in this thesis is provided in Chapter 5 and the materials and methods used in evaluating this work is reported in Chapter 6.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry