Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.659681
Title: Flavocytochrome b2 : investigating structure and function
Author: Mowat, Christopher G.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2000
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Abstract:
Flavocytochrome b2 from Saccharomyces cerevisiae is an L-lactate:cytochrome c oxidoreductase. The crystal structure of this homotetrameric enzyme has been solved to 2.4 A (Xia and Mathews, 1990), and many aspects of its function have been investigated by the construction and kinetic analysis of site-directed mutant forms. From inspection of the crystal structure, Arg289 can be seen to interact with Arg376 which is a key residue for formation of the Michaelis complex. In addition Arg289 also forms a hydrogen bond with a water molecule which is itself hydrogen-bonded to a heme propionate. Substitution of this arginine by lysine results in an enzyme with altered kinetic properties. The Arg289(r)Lys mutant flavocytochrome b2 (R289K-b2) is shown to have a decreased kcat for ferricyanide-dependent L-lactate dehydrogenation (33.0 ± 3.9 vs. 400 ± 10 s-1 for the wild-type enzyme) with an increased value of kcat for L-lactate (1.40 ± 0.20 mM vs. 0.49 ± 0.05 mM for the wild-type enzyme). With cytochrome c as electron acceptor, the kcat for L-lactate dehydrogenation by R289K-b2 is also decreased by an order of magnitude relative to wild-type enzyme. Pre-steady state kinetics confirm that the rate constant for FMN reduction by L-lactate is significantly decreased (19.6 ± 1.7 s-1 cf. 604 ± 60 s-1 in the wild-type enzyme), leading to the conclusion that substitution of Arg289 with lysine has caused FMN reduction to become the rate-determining step of the catalytic cycle. This is contrary to the situation for the wild-type enzyme, where transfer of an electron from FMN semiquinone to the heme is rate limiting (Daff et al., 1996a). It is thought that this altered behaviour may be due to structural changes at the active site which affect substrate binding and dehydrogenation. Ligand-binding properties of R289K-b2 were investigated by a series of inhibition experiments which showed significant changes from wild-type behaviour. This was particularly evident with pyruvate, the product of the reaction which displayed a different type of inhibition in R289K-b2. The crystal structure of R289K-b2 was solved to 2.75 A resolution, and this revealed changes in the position of some active site residues, in particular Arg376, a residue which is important in substrate binding.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.659681  DOI: Not available
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