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 (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 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 with holoBCCP87 as substrate is 2000-fold greater than with biotin as substrate.
Blanchard
C Z
CZ
Division of Biochemistry, Louisiana State University, Baton Rouge, Louisiana 70803, USA.
Chapman-Smith
A
A
Wallace
J C
JC
Waldrop
G L
GL
D000103:Acetyl-CoA Carboxylase / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism*; D019878:Carboxyl and Carbamoyl Transferases / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D004926:Escherichia coli; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*
D002352:Carrier Proteins; D010446:Peptide Fragments; C108671:biotin carboxyl carrier protein (70-156); D001710:Biotin; D000255:Adenosine Triphosphate; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase; D019731:Carbon-Nitrogen Ligases; C019791:biotin carboxylase; D000103:Acetyl-CoA Carboxylase