Flavocytochrome <i>b<sub>2</sub></i> from <i>Saccharomyces cerevisiae</i> is an L-lactate:cytochrome <i>c</i> 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 <i>b<sub>2</sub></i> (R289K-<i>b<sub>2</sub></i>) is shown to have a decreased <i>k<sub>cat</sub></i> for ferricyanide-dependent L-lactate dehydrogenation (33.0 ± 3.9 <i>vs</i>. 400 ± 10 s<sup>-1</sup> for the wild-type enzyme) with an increased value of <i>k<sub>cat</sub></i> for L-lactate (1.40 ± 0.20 mM <i>vs</i>. 0.49 ± 0.05 mM for the wild-type enzyme). With cytochrome <i>c</i> as electron acceptor, the <i>k<sub>cat</sub></i> for L-lactate dehydrogenation by R289K-<i>b<sub>2</sub></i> 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<sup>-1 </sup>cf. 604 ± 60 s<sup>-1 </sup>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 <i>et al.</i>, 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-<i>b<sub>2</sub></i> 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-<i>b<sub>2</sub></i>. The crystal structure of R289K-<i>b<sub>2</sub></i> 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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:659681 |
| Date | January 2000 |
| Creators | Mowat, Christopher G. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12127 |
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