PURIFICATION AND PROPERTIES OF D-AROMATIC LACTATE-DEHYDROGENASE AN ENZYME INVOLVED IN THE CATABOLISM OF THE AROMATIC-AMINO-ACIDS OF CANDIDA-MALTOSA

A D-aromatic lactate dehydrogenase was homogeneously purified from Candida maltosa by a procedure including DEAE-cellulose chromatography, ammonium sulfate fractionation, and affinity chromatography on 5'' AMP-Sepharose 4B. The enzyme was found to have a molecular weight of 250,000-280,000 as determined gy gel sieving and glycerol density gradient centrifugation. It is composed of four subunits of Mr 68,000 as shown by gel electrophoresis. The enzyme activity was dependent on the addition of Mn2+ ions (Km = 10 .mu.M). Arrhenius activation energy is 72 kJ/mole. The optimum pH for the reduction reaction and oxidation reaction are 6.5 and 9.5, respectively. The Michaelis constants in both directions were determined to be as follows: p-hydroxyphenylpyruvate 44 .mu.M;phenylpyruvate, 44 .mu.M;indolepyruvate, 67 .mu.M: NADH, 7.7 .mu.M; NADPH, 8.6 .mu.M: p-hydroxy phenyllactate, 76 .mu.M: phenyllactate, 83 .mu.M; indolelactate, 94 .mu.M; NAD+, 93 .mu.M; NADP+, 21 .mu.M.