The lipoic acid residues of the pyruvate dehydrogenase multienzyme complex from Escherichia coli have been modified with radioactive N-ethylmaleimide, N-(3-pyrene)maleimide, and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM). The number of moles of label incorporated per 4.6 × 106 molecular weight is 47 with N-ethylmaleimide, 48 with N-(3-pyrene)maleimide, and 40 with DDPM. The last two
... [Show full abstract] numbers are less precise than the first because of uncertainties in the extinction coefficients of the enzyme bound labels. The overall activity of the enzyme is abolished by modification of the lipoic acid, but the enzymatic activities of pyruvate dehydrogenase and dihydrolipoyl dehydrogenase are unaltered. Direct binding measurements with 1,N6-etheno-CoA indicate that the number of CoA specific sites of the dihydrolipoyl transacetylase is unaltered when the lipoic acid is modified with DDPM, but the dissociation constant increases about a factor of four to 218 μM(0.02 M potassium phosphate, pH 7.0, 5°C). This suggests that lipoic acid interacts with the catalytic site of the transacetylase enzyme. Fluorescence lifetimes were used to measure fluorescence energy transfer within the enzyme complex using the following energy donors: thiochrome diphosphate, bound to the catalytic site of the pyruvate dehydrogenase enzyme; 8-anilino-1-naphthalenesulfonate, bound to the acetyl-CoA regulatory site on the pyruvate dehydrogenase enzyme; or N-(3-pyrene)-maleimide, bound to the lipoic acid on the dihydrolipoyl transacetylase enzyme. The energy acceptors were DDPM, bound to the lipoic acid, or FAD, bound to the dihydrolipoyl dehydrogenase enzyme. No energy transfer was observed between the modified lipoic acid groups and the labeled sites on the pyruvate dehydrogenase or the dihydrolipoyl dehydrogenase. Therefore, the distance between the lipoic acid and these sites must be greater than 40 Å assuming the emission and absorption dipoles are randomly oriented. These results and others previously reported suggest that the simple mechanism of a single lipoic acid rotating between the catalytic sites of the three enzymes is unlikely. An alternative mechanism consistent with existing data is that two or more lipoic acids are used to transfer the intermediates between the three catalytic sites of functionally coupled enzymes in a single catalytic cycle.