Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.657755
Title: Kinetic and spectroscopic studies of flavocytochrome b2
Author: Miles, Caroline S.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1992
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Abstract:
Flavocytochrome b2 (b2) from baker's yeast catalyses the two electron oxidation of L(&43 )lactate to pyruvate and subsequent reduction of cytochrome c. The enzyme is a tetramer of identical subunits, each of which consists of two functionally distinct domains; a flavodehydrogenase domain containing flavin mononucleotide (FMN) and a cytochrome domain containing protoheme IX. The work in this thesis describes the investigation of the roles of specific amino acid residues, carried out through the kinetic and spectroscopic characterisation of site-directed mutant forms of b2. Tyr143 is an active site residue which lies between the flavodehydrogenase and cytochrome domains of b2. Its role in the catalytic cycle was examined by replacement of this residue with phenylalanine. The most significant effect of this mutation was a change in the rate-limiting step of catalysis. In the wild-type enzyme, this is abstraction of the lactate C2-H. In the mutant enzyme, interdomain electron transfer between the flavin and heme prosthetic groups is now the slowest step. The rate of heme reduction by lactate, determined using the stopped-flow method, is decreased by > 20-fold from 445 ± 50 s^-1 in the wild type enzyme to 21 ± 2 s-1 in the mutant enzyme. Decreases in kinetic isotope effects seen with [2-2H]lactate for the mutant enzyme compared to the wild-type, both for flavin and for heme reduction, also provide support for a change in the nature of the rate-limiting step. Other kinetic parameters are all consistent with the mutation having a dramatic effect on inter domain electron transfer. It therefore appears that Tyr143 plays a key role in facilitating electron transfer between the flavin and heme groups. Further studies on the role of Tyr143 were carried out by replacing this residue with glutamine. Kinetic results show that the enzyme is a poor lactate dehydrogenase. Furthermore, the lactate KM has increased by > 100-fold from 0.5mM in the wild-type enzyme to -65mM in the mutant enzyme. These and other kinetic results suggest that lactate to flavin electron transfer is the slowest step in catalysis, due to conformational effects at the active site. This is consistent with circular dichroism studies which show that a significant flavin/aromatic residue interaction has been lost. Two other interdomain residues, Phe325 and Tyr97, were investigated by substitution with alanine and tyrosine respectively. Although kinetic results indicate that Tyr97 is not of great functional significance, it is shown that Phe325 contributes towards domain/domain integrity and recognition.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.657755  DOI: Not available
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