Purification and characterisation of D-amino acid aminotransferase from Rhizobium japonicum.

Rhizobium japonicum has D-amino acid aminotransferase and alanine racemase activities. The D-amino-acid aminotransferase has been partially purified and characterized. This enzyme has a broad specificity and is very active with D-alpha-aminobutyrate and D-aspartate as well as D-alanine and D-glutamate. The stereospecificity of the enzyme for D-amino acids was apparently absolute with respect to product inhibition, pyridoxamine formation as well as catalytic activity. The apparent molecular weight was 58,000 and the pH optimum was 7.8-7.9. The equilibrium constant in the direction of D-glutamate formation was 1.9. Initial-velocity kinetic studies indicate the enzyme acts by a ping-pong mechanism. The dissociation constant for pyridoxal phosphate and the Michaelis constants (+/- standard errors) for D-alanine and 2-oxoglutarate were determined to be 0.51 +/- 0.06 micrometer, and 2.13 +/- 0.18 and 0.058 +/- 0.005 mM respectively. The enzyme is moderately inhibited (30%) by 4 mM p-chloromercuribenzoate.