A general NMR method for the assay of racemase activity with optically active or optically inactive substrates

A rapid and sensitive method is described for determining the activity of alanine racemase (5.1.1.1) with its substrates. Alanine racemase from Bacillus subtilis catalyzes the exchange of the α hydrogen of l and d alanine, a racemic mixture of alanine as well as both the α hydrogens of glycine with D₂O. The rate of exchange which can be followed by NMR spectroscopy is proportional to the enzyme concentration. This assay has been used to test a wide variety of possible substrates and thus determine the specificity of the enzyme.