Rotational diffusion studies of the lipoyl domain of 2-oxoacid dehydrogenase multienzyme complexes.
Rotational mobility of the lipoyl domain of a number of 2-oxoacid dehydrogenase complexes was investigated by transient dichroism after the domain had been specifically labeled with the triplet probe eosin-5-maleimide. Complexes investigated included pyruvate dehydrogenase complexes from Bacillus stearothermophilus, ox heart, and Escherichia coli (in which the E2 component had been genetically engineered to contain one lipoyl domain) and 2-oxoglutarate dehydrogenase complexes from ox heart and E. coli. Measurements were also performed with ox heart pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes specifically labeled on E1. Anisotropy decays were recorded in glycerol-buffer solutions of varying viscosity and at different temperatures. For E2-labeled complexes, the decays were found to be multiexponential, and the fastest correlation time was considerably shorter than expected for tumbling of the whole complex. This fast correlation time was absent from E1-labeled complexes and was assigned to independent motion of the lipoyl domain. Plots of the fast correlation time against eta/T showed a surprisingly weak dependence on viscosity and extrapolated to a time of 30-40 microseconds at zero viscosity. To explain this result, a model is proposed in which the lipoyl domain is in equilibrium between "free" and bound states. The time of 30-40 microseconds is shown to correspond to 1/koff, where koff is the rate constant for dissociation of the domain from binding sites on the complex. This dissociation phenomenon only contributes to the anisotropy decay when the viscosity of the solution is sufficiently high to slow the tumbling of the whole complex to times that are long in comparison to 1/koff.
Department of Chemistry and Biological Chemistry, University of Essex, Wivenhoe Park, Colchester, U.K.
J P
JP
Harrison
I E
IE
Morrison
R J
RJ
Cherry
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D004058:Diffusion; D004801:Eosine Yellowish-(YS); D007658:Ketone Oxidoreductases; D015394:Molecular Structure; D009097:Multienzyme Complexes; D011768:Pyruvate Dehydrogenase Complex; D012399:Rotation; D008063:Thioctic Acid
D009097:Multienzyme Complexes; D011768:Pyruvate Dehydrogenase Complex; D008063:Thioctic Acid; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D004801:Eosine Yellowish-(YS)