Analysis of the biotin-binding site on acetyl-CoA carboxylase from rat (original) (raw)
Related papers
Biochemical and Biophysical Research Communications, 1999
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 reactionintermediate 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.
Biotin Attachment Domain-Containing Proteins Irreversibly Inhibit Acetyl CoA Carboxylase
Plant physiology, 2018
The first committed step in fatty acid synthesis is mediated by acetyl-CoA carboxylase (ACCase), a biotin-dependent enzyme that carboxylates acetyl-CoA to produce malonyl-CoA. ACCase can be feedback regulated by short-term or long-term exposure to fatty acids in the form of Tween 80 (predominantly containing oleic acid), which results in reversible or irreversible ACCase inhibition, respectively. Biotin attachment domain-containing (BADC) proteins are inactive analogs of biotin carboxyl transfer proteins that lack biotin, and their incorporation into ACCase down-regulates its activity by displacing active (biotin-containing) biotin carboxyltransferase protein subunits. Arabidopsis () lines containing T-DNA insertions in , , and were used to generate and double mutants. The mutant exhibited normal growth and development; however, ACCase activity was 26% higher in and its seeds contained 30.1% more fatty acids and 32.6% more triacylgycerol relative to wild-type plants. To assess wheth...
Journal of Biological Chemistry, 1998
Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report the overexpression of the genes for the carboxyltransferase component is described. The steady-state kinetics of the recombinant carboxyltransferase are characterized in the reverse direction, in which malonyl-CoA reacts with biocytin to form acetyl-CoA and carboxybiocytin. The initial velocity patterns indicated that the kinetic mechanism is equilibrium-ordered with malonyl-CoA binding before biocytin and the binding of malonyl-CoA to carboxyltransferase at equilibrium. The biotin analogs, desthiobiotin and 2-imidazolidone, inhibited carboxyltransferase. Both analogs exhibited parabolic noncompetitive inhibition, which means that two molecules of inhibitor bind to the enzyme. The pH dependence for both the maximum velocity (V) and the (V/K) biocytin parameters decreased at low pH. A single ionizing group on the enzyme with a pK of 6.2 or lower in the (V/K) biocytin profile and 7.5 in the V profile must be unprotonated for catalysis. Carboxyltransferase was inactivated by N-ethylmaleimide, whereas malonyl-CoA protected against inactivation. This suggests that a thiol in or near the active site is needed for catalysis. The rate of inactivation of carboxyltransferase by N-ethylmaleimide decreased with decreasing pH and indicated that the pK of the sulfhydryl group had a pK value of 7.3. It is proposed that the thiolate ion of a cysteine acts as a catalytic base to remove the N1 proton of biocytin.