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

Abstract

Poly(ethyleneglyco1)-bound NAD (PEG-NAD) was covalently linked to Thermus thermophilus malate dehydrogendse 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 11, MDH-PEG-NAD 111 and MDH-PEG-NAD IV. The average numbers of NAD moieties covalently bound per subunit of MDH-PEG-NAD I, MDH-PEG-NAD 11, MDH-PEG-NAD I11 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/polydcrylamide gel electrophoresis. 60 -80% bound NAD moiety of these preparations of MDH-PEG-NAD was reduced by the enzyme moiety in the presence of Lmalate, 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 , 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-l. 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.A covalently linked dehydrogenase-NAD complex is an attractive catalytic unit if the bound NAD can be used by other enzymes as well as by itself. Such a complex serves as an NAD(H)-regeneration unit in enzyme reactors and also provides many interesting questions about the effects of fixing a readily dissociable coenzyme in the vicinity of an enzyme. Several efforts have been made for this purpose [l -31 but only the alcohol-dehydrogenase -NAD complex, reported by MAnsson et al. [2], has been clearly demonstrated to have covalently bound NAD. As for the complex, recycling of the bound NAD by lactate or malate dehydrogenase [4] and kinetic properties of the alcohol dehydrogenase activity of the complex [5] have also been investigated.We have prepared poly(ethyleneglyco1)-bound NAD (PEG-NAD) as a macromolecular NAD derivative for enzyme reactors [6, 71. PEG-NAD has a unique structure in that one NAD molecule and one amino group are linked with Correspondence to I. Urabe,

Synthesis and application of water-soluble macromolecular derivatives of the redox coenzymes NAD(H), NADP(H) and FAD

“…One of the striking properties of dehydrogenase-NAD complexes is the intramolecular character of the reaction between the NAD moiety and the enzyme moiety, unlike the intermolecular reaction between free NAD and enzyme. Eguchi et al 97) and Nakamura et al 9s), converting L-malate or glucose in the presence of a chemical redox system for NAD recycling and monitoring the reaction, proved that the rate constants for malate dehydrogenase-PEG (M r 3,000)-NAD and glucose detrydrogenase-PEG (M r 3,000)-NAD complexes (spacer~ nm) were independent of the concentration of the complexes, showing first-order kinetics. In contrast, the reshlts of control experiments with free NAD and enzyme at a fixed ratio pointed to second-order kinetics.…”

Preparation and kinetic properties of 5‐ethylphenazine–lactate‐dehydrogenase–NAD+ conjugate, a semisynthetic lactate oxidase showing a hide‐and‐seek effect

“…The activity of the conjugate is still lower than that of the native enzyme and increases with the increase in the NAD' concentration, while the activity of the native enzyme at 68.8 mM NAD' is the same as that at 1.6 mM NAD'. This means that the conjugate has much larger apparent K, value for [8]. These larger K , values for exogenous NAD' of the conjugates are considered to be due to competitive inhibition by the bound NADH moiety.…”

Construction of Enzyme-Cofactor/Mediator Conjugates for Enhanced in Vitro Bioelectricity Generation

“…PEGylation, with the remarkable therapeutic and biotechnological potential of peptides and proteins, is a process of attaching polyethylene glycol (PEG) chains to molecules of interest . PEGylated NAD­(P) + derivatives have been developed for a long time to investigate cofactor regeneration for dehydrogenase-catalyzed reactions. , For example, NAD + -PEG has been reported to covalently bind to GDH or malate dehydrogenase and form enzyme-PEG-NAD + conjugates with dramatically accelerated reaction rates. Recently, Kim et al has demonstrated a boosted biohydrogen production rate using NAD + -conjugated dehydrogenases .…”

“…23 PEGylated NAD(P) + derivatives have been developed for a long time to investigate cofactor regeneration for dehydrogenase-catalyzed reactions. 24,25 NAD + -PEG has been reported to covalently bind to GDH 26 or malate dehydrogenase 27 and form enzyme-PEG-NAD + conjugates with dramatically accelerated reaction rates. Recently, Kim et al has demonstrated a boosted biohydrogen production rate using NAD + -conjugated dehydrogenases.…”