Affinity labeling of chicken liver fatty acid synthase with chloroacetyl-CoA and bromopyruvate.
Fatty acid synthase of chicken liver is inactivated rapidly and irreversibly by incubation with chloroacetyl-CoA or with bromopyruvate. Inactivation by both reagents follows saturation kinetics, indicating the formation of an E ... I complex (dissociation constants of 0.36 microM for chloroacetyl-CoA and 31 microM for bromopyruvate) prior to alkylation. The limiting rate constants are 0.15 s-1 for bromopyruvate and 0.041 s-1 for chloroacetyl-CoA. Inactivation by both reagents is protected by NADPH and 200 mM KCl, and by saturating amounts of thioester substrates which reduced the limiting rate constants 6.5-30-fold. Active-site-directed reaction of chloroacetyl-CoA is supported by the ability of this compound to form a kinetically viable complex with the enzyme as competitive inhibitor of acetyl-CoA. Chloroacetyl-CoA interacts initially at the CoA binding pocket, since the nucleotide afforded competitive protection of inactivation and caused a large decrease in its affinity. Subsequently, the phosphopantetheine prosthetic group is alkylated. Evidence is presented to show that bromopyruvate competes with chloroacetyl-CoA for the same target site.
Tian
W X
WX
State University of New York Health Science Center, Syracuse.
Wang
Y S
YS
Hsu
R Y
RY
D000105:Acetyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels; D000478:Alkylation; D000818:Animals; D001667:Binding, Competitive; D002645:Chickens; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009249:NADP / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011773:Pyruvates / Q000378:metabolism / Q000494:pharmacology*
D000345:Affinity Labels; D011773:Pyruvates; C026772:chloroacetyl coenzyme A; D009249:NADP; C017092:bromopyruvate; D011189:Potassium Chloride; D000105:Acetyl Coenzyme A; D064429:Fatty Acid Synthases