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

J Biol Chem. 1999 Nov 5;274(45):31767-9. doi: 10.1074/jbc.274.45.31767.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetyl-CoA Carboxylase / metabolism*
  • Adenosine Triphosphate / biosynthesis
  • Binding Sites
  • Biotin / metabolism*
  • Carbon-Nitrogen Ligases / metabolism*
  • Carboxyl and Carbamoyl Transferases / metabolism*
  • Carrier Proteins / chemistry
  • Carrier Proteins / metabolism*
  • Catalysis
  • Enzyme Activation
  • Escherichia coli
  • Kinetics
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism*

Substances

  • Carrier Proteins
  • Peptide Fragments
  • biotin carboxyl carrier protein (70-156)
  • Biotin
  • Adenosine Triphosphate
  • Carboxyl and Carbamoyl Transferases
  • Methylmalonyl-CoA carboxytransferase
  • Carbon-Nitrogen Ligases
  • biotin carboxylase
  • Acetyl-CoA Carboxylase