Flavocytochrome <i>c<sub>3</sub> </i>is a soluble , periplasmic, fumarate reductase from <i>Shewanella frigidimarina </i>which consists of three domains. This thesis reports an investigation into the enzyme mechanism by site directed mutagenesis. Fumarate is bound in the active site via important interactions with the side chains of His504, His365, Thr377 and Arg544. The substitution of Thr377 by alanine causes a 13-fold decrease in k<sub>cat</sub> and a 27-fold increase in K<sub>M, </sub>consistent with a role purely in substrate binding. Substituting Arg544 with methionine dramatically lowers the k<sub>cat</sub> by 10<sup>4</sup>-fold and raises the K<sub>M</sub> 29-fold. This residue is involved in substrate binding but is likely to have an additional role polarising the substrate molecule, for hydride attack. A structural sodium ion is located close to both the active site and the FAD tail group. It is bound in approximately octahedral geometry by five backbone carbonyls (Thr506, Met507, Gly508, Thr536 and Glu534) and a water molecule. Substituting Glu534 results in an inability to retain FAD. His505 hydrogen bonds to the water molecule ligating the sodium and is also next to His504, which is one of the substrate binding residues. The pH-activity profile of wild-type fits to a single pK<sub>a</sub> value of 7.5 ± 0.1 which is attributed to His504 stabilising the build up of charge in the reaction intermediate. This pK<sub>a</sub> is raised to 8.2 ± 0.1 by the substitution H505Y and to 9.0 ± 0.2 by H505A. The k<sub>cat</sub> value is lowered 2-fold and 20-fold for these mutants respectively. In the mutant enzymes His504 has become a weaker acid and is less able to enhance the rate at low pH. The role of His505 may be to moderate the effect of the negative charge of Glu534 on His504. The active site acid, Arg402, has a dual role as both a Lewis acid (stabilising the build up of charge after hydride transfer) and a Brønsted acid (delivering a proton to the substrate C3). The structure of the mutant enzyme R402A revealed a water molecule at the active site (Mowat; 2001), but this is too far from the substrate C3 to act as the acid catalyst and the enzyme is completely inactive (Doherty; 2000). The double mutant Q363F/R402A, however, is active but at a level 10<sup>4</sup>-fold lower than wild-type. The structure shows that the water molecule is now close enough to the substrate C3 to act as an acid catalyst. Wild-type has an overall solvent isotope effect k<sub>H</sub>/k<sub>D</sub>, of 8.2 ± 0.4 and its proton inventory fits to a model for multiple exchangeable hydrogenic sites, consistent with a complex transition state involving a proton pathway. Q363F/R402A has double the solvent isotope effect of wild-type and the proton inventory indicates that the transition state remains complex. So the active site water is trapped and requires reprotonation by the proton pathway. Substituting Arg402 by glutamine lowers the k<sub>cat</sub> 10<sup>5</sup>-fold. Not only is glutamine a poor Lewis acid but the structure shows that the shorter side chain results in an increased proton transfer distance. The mutant R402F is completely inactive as phenylalanine is unable to protonate the substrate.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:660311 |
| Date | January 2002 |
| Creators | Pankhurst, Katherine L. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/15582 |
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