Haruhiko Sakuraba

Purification and characterization of malate dehydrogenase from the phototrophic bacterium, Rhodopseudomonas capsulata

Abstract Malate dehydrogenase ( l -malate:NAD + oxidoreductase, EC 1.1.1.37) has been purified about 480-fold from crude extract of the facultative phototrophic bacterium, Rhodopseudomonas capsulata by only two purification steps, involving Red-Sepharose affinity chromatography. The enzyme has a molecular mass of about 80 kDa and consists of two subunits with identical molecular mass (35 kDa). The enzyme is susceptible to heat inactivation and loses its activity completely upon incubation at 40°C for 10 min. Addition of NAD + , NADH and oxaloacetate, but not l -malate, to the enzyme solution stabilized the enzyme. The enzyme catalyzes exclusively the oxidation of l -malate, and the reduction of oxaloacetate and ketomalonate in the presence of NAD + and NADH, respectively, as the coenzyme. The pH optima are around 9.5 for the l -malate oxidation, and 7.75–8.5 and 4.3–7.0 for the reduction of oxaloacetate and ketomalonate, respectively. The K m values were determined to be 2.1 mM for l -malate, 48 μM for NAD + , 85 μM for oxaloacetate, 25 μM for NADH and 2.2 mM for ketomalonate. Initial velocity and product inhibition patterns of the enzyme reactions indicate a random binding of the substrates, NAD + and l -malate, to the enzyme and a sequential release of the products: NADH is the last product released from the enzyme in the l -malate oxidation.