The enzyme succinyl coenzyme A synthetase catalyzes the formation of succinyl coenzyme A (CoA) from succinate, CoA, and a nucleoside triphosphate. The enzyme has been labeled with dansyl chloride in both the α and β subunits to give an almost fully active fluorescent conjugate. Addition of CoA at 1 mM caused the polarization of the conjugate to fall from ~0.275 to ~0.145. Control studies indicated that this result was not due to effects such as dissociation of the protein, release of bound label, or changes in the lifetime of the bound label. The other substrates, ATP-Mg2+, succinate, or ADP·Mg2+, gave a polarization decrease when added to the conjugate, but to a much smaller degree compared to the effect of CoA. For example, the maximum decrease with the other substrates was observed with ADP·Mg2+ where the polarization only fell to 0.245 at 5 mM ADP·Mg2+. Comparison with model compounds indicates that the effect of CoA is specific. Analysis of the polarization data gives rotational relaxation times for the conjugate which are smaller than that expected for the protein considered as a sphere, in both the presence and absence of CoA. Perrin-Weber plots in the presence of CoA are concave to the T/η axis. These results suggest that the dansyl label can be covalently bound to a flexible site on succinyl-CoA synthetase and the flexibility is greatly increased on CoA binding. These results are compatible with a model for succinyl-CoA synthetase in which CoA binding induces a conformational change in the active site and help explain previous kinetic observations that the presence of CoA can affect the rates and partial reactions involving the other enzyme substrates.
|Original language||English (US)|
|Number of pages||6|
|State||Published - 1982|
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