Covalent linking of poly(ethyleneglycol)‐bound NAD with Thermus thermophilus malate dehydrogenase

Poly(ethyleneglycol)‐bound NAD (PEG‐NAD) was covalently linked to Thermus thermophilus malate dehydrogenase with a bifunctional reagent, 3,3′‐(1,6‐dioxo‐1,6‐hexanediyl)bis‐2‐thiazolidinethione. The covalently linked malate‐dehydrogenase–PEG–NAD complex (MDH‐PEG‐NAD) was purified by DEAE‐Sephadex column chromatography to remove unbound PEG‐NAD, and fractionated by blue‐Sepharose column chromatography into four preparations: MDH‐PEG‐NAD I, MDH‐PEG‐NAD II, MDH‐PEG‐NAD III and MDH‐PEG‐NAD IV. The average numbers of NAD moieties covalently bound per subunit of MDH‐PEG‐NAD I, MDH‐PEG‐NAD II, MDH‐PEG‐NAD III and MDH‐PEG‐NAD IV were 1.2, 1.2, 0.8 and 0.5, respectively, and the values were confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. 60–80% bound NAD moiety of these preparations of MDH‐PEG‐NAD was reduced by the enzyme moiety in the presence of L‐malate, and the specific activity of the enzyme moiety of the preparations was more than 80% that of the native enzyme. MDH‐PEG‐NAD I has the following properties. The K m value for exogeneous NAD is three times that of the native enzyme. The coenzyme activity of its NAD moiety is 20–40% that of native NAD for alcohol and lactate dehydrogenases. The complex catalyzes the oxidation of L‐malate in the presence of the redox system of 5‐ethylphenazinium ethyl sulfate and a tetrazolium salt with a rate constant of 0.11 s −1 . The coenzyme moiety of the complex can also be recycled by coupled reactions of the active site of the same complex and alcohol dehydrogenase. These results indicate that MDH‐PEG‐NAD works as an NAD(H)‐regeneration unit for coupled reactions.