Covalent linking of poly(ethyleneglycol)-bound NAD with Thermus thermophilus malate dehydrogenase. NAD(H)-regeneration unit for a coupled second-enzyme reaction.

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 Km value for exogenous 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.