Importance of methionine residues in the enzymatic carboxylation of biotin-containing peptides representing the local biotinyl site of E. coli acetyl-CoA carboxylase

Abstract

A biotin-containing hexapeptide Ac-Glu-Ala-Met-Bct-Met-Met (1) that represents the local biotin-containing site of Escherichia coli acetyl-CoA carboxylase has been prepared by the solid phase method. Peptide 1 is carboxylated by the biotin carboxylase subunit dimer of E. coli acetyl-CoA carboxylase with the following kinetic parameters; Km 12 mM, Vmax 2.8 microM X min-1. These compare with the parameters for biotin of Km 214 mM and Vmax 28 microM X min -1. Hence, the overall reactivity (Vmax/Km) of 1 is 1.8 times greater than that of free biotin. When all methionines in 1 are replaced by alanine, the resulting peptide (2) retains a similar binding ability but with a much decreased Vmax. It was also found that peptide 3, which carries an N epsilon-benzyloxycarbonyllysine in place of biocytin in 1, decreases the Km of biotin threefold.