J Biol Chem, Vol. 274, Issue 45, 31767-31769, November 5, 1999

COMMUNICATION
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

From the Division of Biochemistry and Molecular Biology, Louisiana State University, Baton Rouge, Louisiana 70803 and the Department of Biochemistry, University of Adelaide, Adelaide, South Australia 5005, Australia

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 (holoBCCP87) 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 min¹. 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 min¹. The V/K or catalytic efficiency of carboxyltransferase with holoBCCP87 as substrate is 2000-fold greater than with biotin as substrate.