Kinetic Analysis of L-Carnosine Formation by β-Aminopeptidases

Abstract

The β,α-dipeptide L-carnosine occurs in high concentrations in long-lived innervated mammalian tissues and is widely sold as a food additive. On a large scale L-carnosine is produced by chemical synthesis procedures. We have established two aqueous enzymatic reaction systems for the preparation of L-carnosine using the dissolved bacterial β-aminopeptidases DmpA from Ochrobactrum anthropi and BapA from Sphingosinicella xenopeptidilytica as catalysts and investigated the kinetics of the enzyme-catalyzed peptide couplings. DmpA catalyzed the formation of L-carnosine from C-terminally activated β-alanine derivatives (acyl donor) and L-histidine (acyl acceptor) in an aqueous reaction mixture at pH 10 with high catalytic rates (V_max=19.2 μmol min⁻¹ per mg of protein, k_cat=12.9 s⁻¹), whereas V_max in the BapA-catalyzed coupling reaction remained below 1.4 μmol min⁻¹ per mg of protein (k_cat=0.87 s⁻¹). Although the equilibrium of this reaction lies on the side of the hydrolysis products, the reaction is under kinetic control and L-carnosine temporarily accumulated to concentrations that correspond to yields of more than 50% with respect to the employed acyl donor. However, competing nucleophiles caused unwanted hydrolysis and coupling reactions that led to decreased product yield and to formation of various peptidic by-products. The substitution of L-histidine for L-histidine methyl ester as acyl acceptor shifted the pK_a of the amino functionality from 9.25 to 6.97, which caused a drastic reduction in the amount of coupling by-products in an aqueous reaction system at pH 8.