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Title: The kinetic characterisation of Rhodotorula graminis L-mandelate dehydrogenase
Author: Sinclair, Rhona
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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The recombinant form of the R. graminis L-mdh has been overexpressed in E. coli 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-1) and with is physiological electron acceptor, cytochrome c (225 ± 15 s-1). In both cases the kcat values were similar to those previously obtained with L-ldh with L-lactate as substrate. The individual electron transfer steps in the catalytic cycle of L-mdh were also measured using stopped-flow spectrophotometry. The flavin reduction rate (280 ± 20 s-1) appeared to be half that observed for the flavin reduction value obtained with L-ldh (604 ± 60s-1). 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-1) was similar to that observed with L-ldh (445 ± 50 s-1). This intra-molecular electron-transfer step is thought to occur so rapidly in L-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 L-mdh (-120 mV and -10mV respectively) compared to the values previously obtained for L-ldh (-78 mV and -17mV) confirms that there is a larger driving force between the two prosthetic groups of L-mdh. The third step in the catalytic cycle of L-mdh is the inter-molecular transfer step from the haem group to cytochrome c. Stopped-flow studies on the cytochrome c reductase activity of L-mdh yielded a second order rate constant of 41.5 ± 2 μM-1s-1, which represents the rate constant for cytochrome c association. The corresponding value with L-ldh is 34.8 ± 0.9 μM-1 s-1.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available