Analysis of the biotin-binding site on acetyl-CoA carboxylase from rat |
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Authors: | D H Bai T W Moon F López-Casillas P C Andrews K H Kim |
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Affiliation: | Department of Biochemistry, Purdue University, West Lafayette, IN 47907. |
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Abstract: | 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. |
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