Preparation and kinetic properties of 5-ethylphenazine-poly(ethylene-glycol)-glutamate-dehydrogenase conjugate. A semisynthetic NADH oxidase.
5-Ethylphenazine-poly(ethylene glycol)-glutamate dehydrogenase conjugate (EP(+)-PEG-GluDH) was prepared by linking poly(ethylene glycol)-bound 5-ethylphenazine to glutamate dehydrogenase. The average number of the ethylphenazine moieties bound/enzyme subunit was 0.7. This conjugate is a semisynthetic enzyme having NADH oxidase activity; the ethylphenazine moiety works as a catalytic group, and the coenzyme-binding site of glutamate dehydrogenase works as a substrate-binding site. The effects of the presence of the substrate-binding site near the catalytic group were studied by using EP(+)-PEG-GluDH. Before the preparation of the conjugate, the reactivity of NADH bound in the coenzyme-binding site toward the ethylphenazine moiety was estimated for glutamate and lactate dehydrogenases. The results show that the NADH molecule bound in the site of glutamate dehydrogenase reacts with EP(+)-PEG at a rate of 43% of that of free NADH, but the NADH molecule bound in lactate dehydrogenase does not react with 1-(3-carboxypropyloxy)-5- ethylphenazine. Therefore, glutamate dehydrogenase was used as the substrate-binding site of the semisynthetic NADH oxidase. The results of the kinetic analysis of the activity of EP(+)-PEG-GluDH show that the apparent turnover number of the active site is 0.38 s-1, which corresponds to the apparent intramolecular rate constant of the oxidation of NADH bound in the active site. The apparent effective concentration of bound NADH for the catalytic group of the ethylphenazine moiety is 0.33 mM. This means that the presence of the substrate-binding site near the catalytic group increases the local NADH concentration by at most 0.33 mM, and this is the rate-accelerating effect of the binding site.
Yomo
T
T
Department of Fermentation Technology, Faculty of Engineering, Osaka University, Japan.
Urabe
I
I
Okada
H
H
D001665:Binding Sites; D002384:Catalysis; D007700:Kinetics; D009097:Multienzyme Complexes / Q000138:chemical synthesis* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000138:chemical synthesis* / Q000378:metabolism; D013379:Substrate Specificity
D009097:Multienzyme Complexes; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases