Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.643206
Title: Probing the active site of flavocytochrome P450 BM3
Author: Clark, Jonathan P.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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Abstract:
The self sufficient class III monooxygenase, flavocytochrome P450 BM3, from Bacillus megaterium, is a heme containing redox enzyme that catalyses the hydroxylation of long-chain fatty acids. Flavocytochrome P450 BM3 contains several active-site residues that are highly conserved throughout the P450 superfamily. Of these, a phenylalanine (Phe393) has been shown to modulate the heme reduction potential (Em) across a 200 mV range via interactions with the implicitly conserved heme-ligand cysteine. In addition, a distal threonine (Thr268) has been implicated in a variety of roles including proton donation, oxygen activation and substrate recognition. Substrate binding in P450 BM3 causes a shift in the spin state from low- to high-spin. This change in spin-state is accompanied by a positive shift in the reduction potential (ΔEm[WT + arachidonate (120 μM)] = +138 mV). Substitution of Thr268 by alanine or asparagines causes a significant decrease in the ability of the enzyme to generate the high-spin complex via substrate binding and consequently leads to a decrease in the substrate-induced potential shift (ΔEm[T268A + arachidonate (120 μM)] = +73 mV, ΔEm {T268N + arachidonate (120 μM)] = +9 mV). Rate constants for the first electron transfer and for oxy-ferrous decay were measured by pre-steady state stopped-flow kinetics and found to be almost entirely dependant on the heme reduction potential. More positive reduction potentials lead to enhanced rate constants for heme reduction and more stable oxy-ferrous species. These results suggest an important role for this active-site threonine in substrate recognition and in maintaining an efficiently functioning enzyme.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.643206  DOI: Not available
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