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 μm· min–1. These compare with the parameters for biotin of Km 214 mm and Vmax 28 μm· 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‐benzyloxy carbonyllysine in place of biocytin in 1, decreases the Km of biotin threefold.