FURTHER PURIFICATION AND PROPERTIES OF ADENOSINE TRIPHOSPHATE-DEPENDENT PHENYLALANINE RACEMASE OF BACILLUS BREVIS NAGANO

Phenylalanine racemase of Bacillus brevis NAGANO which produces gramicidin S was purified to a nearly homogeneous state. The purification procedure involved ultracentrifugation, ammonium sulfate fractionation, calcium phosphate gel adsorption, gelfiltration with Sephadex G-150, and chromatography on DEAE-Sephadex A-50. The nearly homogeneous enzyme preparation possessed two enzymatic activities; phenylalanine racemization and phenylalanine activation. The molecular weight of the enzyme was estimated to be 100, 000 by the sucrose density gradient centrifugation method. Activation energy was estimated to be 10.9×103 cal per mole. The Km values for L-phenylalanine and ATP were determined to be 2.0×10-5m and 1.5×10-4m, respectively. The velocity of the D-phenylalanine formation from the L-isomer was markedly affected by the concentration of dithiothreitol and pH, whereas that of L-phenylalanine formation from the D-isomer was less affected. ATP was consumed during the conversion of L-phenylalanine into the D-isomer with the concomitant formation of AMP. AMP as well as PP1 enhanced the enzyme activity several fold. © 1969 BY THE JOURNAL OF BIOCHEMISTRY.