Fluorescent derivatives of the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex.
One sulfhydryl group per polypeptide chain of the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was selectively labeled with N-[P-(2-benzoxazoyl)phenyl]-maleimide (NBM), 4-dimethylamino-4-magnitude of-maleimidostilbene (NSM), and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) in 0.05 M potassium phosphate (pH 7). Modification of the sulfhydryl group did not alter the enzymatic activity or the binding of 8-anilino-1-naphthalenesulfonate (ANS) or thiochrome diphosphate to the enzyme. The fluorescence of the NBM or NSM coupled to the sulfhydryl group on the enzyme was quenched by binding to the enzyme of the substrate pyruvate the coenzyme thiamine diphosphate, the coenzyme analogue thiochrome diphosphate, the regulatory ligands acetyl-CoA, GTP, and phosphoenolpyruvate, and the acetyl-CoA analogue, ANS. Fluorescence energy transfer measurements were carried out for the enzyme-bound donor-acceptor pairs NBM-ANS, NBM-thiochrome diphosphate ANS-DDPM, and thiochrome diphosphate-DDM. The results indicate that the modified sulfhydryl group is more than 40 A from the active site and approximately 49 A from the acetyl-CoA regulatory site. Thus, a conformational change must accompany the binding of ligands to the regulatory and catalytic sites. Anisotropy depolarization measurements with ANS bound on the isolated pyruvate dehydrogenase in 0.05 M potassium phosphate (pH 7.0) suggest that under these conditions the enzyme is dimeric.
D000817:Anilino Naphthalenesulfonates; D004735:Energy Transfer; D004926:Escherichia coli; D008071:Lipoprotein Lipase / Q000097:blood; D008079:Lipoproteins, VLDL / Q000378:metabolism; D008301:Maleimides; D009943:Organophosphorus Compounds; D011743:Pyrimidines; D013831:Thiamine / Q000031:analogs & derivatives; D013844:Thiazoles
D000817:Anilino Naphthalenesulfonates; D008079:Lipoproteins, VLDL; D008301:Maleimides; D009943:Organophosphorus Compounds; D011743:Pyrimidines; D013844:Thiazoles; C009460:thiochrome diphosphate; D008071:Lipoprotein Lipase; D013831:Thiamine