The recombinant form of the <I>R. graminis </I><SUB>L</SUB>-mdh has been overexpressed in <I>E. coli</I> to levels around 14% of the total cell protein. The enzyme was purified to homogeneity after several ion-exchange and gel filtration columns. The steady state turnover of the enzyme was measured with the artificial electron acceptor, potassium ferricyanide (550 ± 25 s<SUP>-1</SUP>) and with is physiological electron acceptor, cytochrome <I>c</I> (225 ± 15 s<SUP>-1</SUP>). In both cases the k<SUB>cat</SUB> values were similar to those previously obtained with <SUB>L</SUB>-ldh with <SUB>L</SUB>-lactate as substrate. The individual electron transfer steps in the catalytic cycle of <SUB>L</SUB>-mdh were also measured using stopped-flow spectrophotometry. The flavin reduction rate (280 ± 20 s<SUP>-1</SUP>) appeared to be half that observed for the flavin reduction value obtained with <SUB>L</SUB>-ldh (604 ± 60s<SUP>-1</SUP>). The next step is an intra-molecular electron transfer step between the reduced flavin and oxidised haem, resulting in flavin semiquinone and reduced haem. The rate observed for the haem reduction (605 ± 50 s<SUP>-1</SUP>) was similar to that observed with <SUB>L</SUB>-ldh (445 ± 50 s<SUP>-1</SUP>). This intra-molecular electron-transfer step is thought to occur so rapidly in <SUB>L</SUB>-mdh, that full reduction of the flavin groups is not observed until after a disproportionation of the flavin semiquinones, generating 2 fully reduced and 2 oxidised flavins, and further reduction by 2 molecules of mandelate. Measurement of the flavin and haem midpoint potentials of <SUB>L</SUB>-mdh (-120 mV and -10mV respectively) compared to the values previously obtained for <SUB>L</SUB>-ldh (-78 mV and -17mV) confirms that there is a larger driving force between the two prosthetic groups of <SUB>L</SUB>-mdh. The third step in the catalytic cycle of <SUB>L</SUB>-mdh is the inter-molecular transfer step from the haem group to cytochrome <I>c. </I> Stopped-flow studies on the cytochrome <I>c</I> reductase activity of <SUB>L</SUB>-mdh yielded a second order rate constant of 41.5 ± 2 μM<SUP>-1</SUP>s<SUP>-1</SUP>, which represents the rate constant for cytochrome <I>c</I> association. The corresponding value with <SUB>L</SUB>-ldh is 34.8 ± 0.9 μM<SUP>-1</SUP> s<SUP>-1</SUP>.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:661967 |
| Date | January 1998 |
| Creators | Sinclair, Rhona |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/14427 |
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