Inhibition of biotin carboxylase by a reaction intermediate analog: implications for the kinetic mechanism.
The first committed step in long-chain fatty acid synthesis is catalyzed by the multienzyme complex acetyl CoA carboxylase. One component of the acetyl CoA carboxylase complex is biotin carboxylase which catalyzes the ATP-dependent carboxylation of biotin. The Escherichia coli form of biotin carboxylase can be isolated from the other components of the acetyl CoA carboxylase complex such that enzymatic activity is retained. The synthesis of a reaction intermediate analog inhibitor of biotin carboxylase has been described recently (Organic Lett. 1, 99-102, 1999). The inhibitor is formed by coupling phosphonoacetic acid to the 1'-N of biotin. In this paper the characterization of the inhibition of biotin carboxylase by this reaction-intermediate analog is described. The analog showed competitive inhibition versus ATP with a slope inhibition constant of 8 mM. Noncompetitive inhibition was found for the analog versus biotin. Phosphonoacetate exhibited competitive inhibition with respect to ATP and noncompetitive inhibition versus bicarbonate. Biotin was found to be a noncompetitive substrate inhibitor of biotin carboxylase. These data suggested that biotin carboxylase had an ordered addition of substrates with ATP binding first followed by bicarbonate and then biotin.
Blanchard
C Z
CZ
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA.
Amspacher
D
D
Strongin
R
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Waldrop
G L
GL
D000255:Adenosine Triphosphate / Q000378:metabolism; D001667:Binding, Competitive; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D019731:Carbon-Nitrogen Ligases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D015394:Molecular Structure; D010746:Phosphonoacetic Acid / Q000494:pharmacology
D004791:Enzyme Inhibitors; D001710:Biotin; D000255:Adenosine Triphosphate; D019731:Carbon-Nitrogen Ligases; C019791:biotin carboxylase; D010746:Phosphonoacetic Acid