Fluorescent derivatives of the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex

Abstract

One SH group/polypeptide chain of the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from E. coli was selectively labeled with N-[p-(2-benzoxazolyl)phenyl]-maleimide (NBM), 4-dimethylamino-4''-maleimidostilbene (NSM) and N-(4-dimethylamino-3,5- dinitrophenyl)maleimide (DDPM) in 0.05 M potassium phosphate (pH 7). Modification of the SH 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 SH group on the enzyme was quenched by binding to the enzyme of the substrate pyruvate the coenzyme thiamine diphosphate, the coenzyme analog thiochrome diphosphate, the regulatory ligands acetyl-CoA, GTP, and phosphoenolpyruvate and the acetyl-CoA analog, 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-DDPM. The modified SH group is probably > 40 .ANG. from the active site and .apprx. 49 .ANG. 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.