The biotin domain peptide from the biotin carboxyl carrier protein of Escherichia coli acetyl-CoA carboxylase causes a marked increase in the catalytic efficiency of biotin carboxylase and carboxyltransferase relative to free biotin

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase activity, a biotin carboxyl carrier protein, and a carboxyltransferase activity. The C-terminal 87 amino acids of the biotin carboxyl carrier protein (BCCP87) form a domain that can be independently expressed, biotinylated, and purified (Chapman-Smith, A., Turner, D. L., Cronan, J. E., Morris, T. W., and Wallace, J, C, (1994) Biochem, J, 302, 881-887), The ability of the biotinylated form of this 87-residue protein (holo BCCP87) to act as a substrate for biotin carboxylase and carboxyltransferase was assessed and compared with the results with free biotin, In the case of biotin carboxylase holoBCCP87 was an excellent substrate with a K-m, of 0.16 +/- 0.05 mM and V-max of 1000.8 +/- 182.0 min(-1). The V/K or catalytic efficiency of biotin carboxylase with holoBCCP87 as substrate was 8000-fold greater than with biotin as substrate. Stimulation of the ATP synthesis reaction of biotin carboxylase where carbamyl phosphate reacted with ADP by holoBCCP87 was 5-fold greater than with an equivalent amount of biotin, The interaction of holoBCCP87 with carboxyltransferase was characterized in the reverse direction where malonyl-CoA reacted with holoBCCP87 to form acetyl-CoA and carboxyholoBCCP87, The K-m for holoBCCP87 was 0.45 +/- 0.07 mM while the V-max was 2031.8 +/- 231.0 min(-1). The V/K or catalytic efficiency of carboxyltransferase.