Amino Acid Racemase Enzyme Assays

Amino acid racemases are enzymes that invert the α-carbon stereochemistry of amino acids (AAs), interconverting amino acids between their L- and D-enantiomers in a reversible reaction. In bacteria, they are known to have catabolic physiological functions but are also involved in the synthesis of many D-AAs, including D-glutamate and D-alanine, which are necessary components of the peptidoglycan layer of the bacterial cell wall. As such, amino acid racemases represent significant targets for the development of bactericidal compounds. Amino acid racemases are also regarded by the biotechnological industry as important catalysts for the production of economically relevant D-AAs. Here, we provide a detailed protocol using high performance liquid chromatography (HPLC) and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, also Marfey’s reagent) for the characterization of novel amino acid racemases. The protocol described here was designed to obtain accurate kinetic parameters (kcat, KM values). Enzyme concentrations and reaction times were optimized so as to minimize the reverse reaction, which can confound results when measuring racemase reactions.