Affinity labeling of chicken liver fatty acid synthase with chloroacetyl-CoA and bromopyruvate.
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| Abstract |
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.
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| Authors | W X Tian, Y S Wang, R Y Hsu |
| Journal | Biochimica et biophysica acta (Biochim Biophys Acta) Vol. 998 Issue 3 Pg. 310-6 (Oct 19 1989) ISSN: 0006-3002 [Print] Netherlands |
| PMID | 2804133 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.) |
| Chemical References |
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