Analysis of the biotin-binding site on acetyl-CoA carboxylase from rat.
The biotin-binding site of acetyl-CoA carboxylase from rat was characterized as to its amino acid sequence and relative position in the enzyme molecule. Biotin binds to the lysyl residue in the tetrapeptide Val-Met-Lys-Met; this tetrapeptide is located in close proximity to the NH2 terminus. In all other biotin-containing enzymes, the conserved tetrapeptide Ala-Met-Lys-Met is the counterpart to that of rat acetyl-CoA carboxylase; and the lysyl residue is 35 residues from the COOH terminus. To examine the significance of these unusual features of the biotinylation site of animal acetyl-CoA carboxylase, cDNA fragments were expressed in a bacterial system and the effects of specific site-directed mutagenesis were examined. Replacement of Val by Ala in the conserved tetrapeptide abolished biotinylation of the expressed protein. However, introduction of a termination codon at residue 36, in such a way that the distance between the lysine on which biotin binds and the COOH-terminal amino acid was 35 residues and the penultimate amino acid was the hydrophobic residue leucine, increased the efficiency of biotinylation, provided a substantial portion of the NH2-terminal peptide was removed.
Bai
D H
DH
Department of Biochemistry, Purdue University, West Lafayette, IN 47907.
Moon
T W
TW
López-Casillas
F
F
Andrews
P C
PC
Kim
K H
KH
D000103:Acetyl-CoA Carboxylase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D008025:Ligases / Q000378:metabolism*; D008239:Lysine / Q000032:analysis; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000302:isolation & purification; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D014633:Valine / Q000032:analysis
D010455:Peptides; D011994:Recombinant Proteins; D001710:Biotin; D004247:DNA; D008025:Ligases; D000103:Acetyl-CoA Carboxylase; D014633:Valine; D008239:Lysine