Inactivation of the dadB Salmonella typhimurium alanine racemase by D and L isomers of .beta.-substituted alanines: kinetics, stoichiometry, active site peptide sequencing, and reaction mechanism

Abstract

The pyridoxal phosphate dependent S. typhimurium dadB alanine racemase was inactivated with D and L-.beta.-fluoroalanine, D- and L-.beta.-chloroalanine and O-acetyl-D-serine. Enzyme inactivation with each isomer of .beta.-chloro[14C]alanine followed by NaBH4 reduction and trypsin digestion afforded a single radiolabeled peptide. In the same manner, NaB3H4-reduced native enzyme gave a single labeled peptide after trypsin digestion. Purification and sequencing of these 3 radioactive peptides revealed them to be a common, unique hexadecapeptide which contained labeled lysine at position 6 in each case. Enzyme which had been inactivated, but not reductively stabilized with NaBH4, released a labile pyridoxal phosphate-inactivator adduct on denaturation. The structure of this adduct suggests that the enzyme was inactivated by trapping the coenzyme in a ternary adduct with inactivator and the active site lysine. Under denaturing conditions, facile .alpha.,.beta.-elimination occurred, releasing the aldol adduct of pyruvate and pyridoxal phosphate. Reduction of the ternary enzyme adduct blocked this elimination pathway. The overall mechanism of racemase inactivation is discussed in light of these results.