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

Abstract

-EJB 91 0860 5-Ethylphenazine -lactate-dehydrogenase -NAD + conjugate (EP+-LDH-NAD') was prepared by linking poly(ethy1ene glycol)-bound Sethylphenazine and poly(ethy1ene glycol)-bound NAD ' to lactate dehydrogenase. The average number of the ethylphenazine moieties bound per molecule of enzyme subunit was 0.46, and that of the NAD+ moieties was 0.32. This conjugate is a semisynthetic enzyme having lactate oxidase activity using oxygen or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as an electron acceptor; to make such conjugates seems to be a general method for artificially converting a dehydrogenase into an oxidase. When the concentration of oxygen or MTT is varied, the oxidase activity fits the Michaelis-Menten equation with the following kinetic constants: for the reaction system with oxygen, the turnover number per subunit is 2.3 min-' and K, for oxygen is 1.91 mM; and for the system with MTT, the turnover number is 0.25 min-' and K, for MTT is 0.076 mM. At the initial steady state of the oxidase reaction, only 2.1% of the NAD' moieties of the conjugate are in the free state (i.e. not bound in the coenzyme-binding site of the lactate dehydrogenase moiety) and the rest are hidden in the coenzyme site; almost all the NAD+ moieties are in the reduced state. The apparent intramolecular rate constant for the reaction between a free NADH moiety and an oxidized ethylphenazine moiety is 2.3 s-' and 2.1 s-' for the systems with oxygen and with MTT, respectively. The apparent effective concentration of the free NADH moiety for the ethylphenazine moiety is 5.5 pM and is much smaller than that (0.34 mM) of the ethylphenazine moiety for the free NADH moiety; this difference is due to the effect of hiding the NADH moiety in the binding site, as the hidden NADH moiety cannot react with the ethylphenazine moiety.We have prepared several types of enzyme-cofactor conjugates by covalently linking 5-ethylphenazine and NAD + to dehydrogenases for the purpose of obtaining kinetic information for designing enzymes and enzyme-like catalysts. At first, we prepared EP'-PEG-GluDH by linking 5-ethylphenazine (EP ') to glutamate dehydrogenase (GluDH) via a longer spacer of poly(ethy1ene glycol) (PEG; M , 3000) [l]. This conjugate worked as a semisynthetic NADH oxidase, using the ethylphenazine moiety as an artificial catalytic group and the coenzyme-binding site of the dehydrogenase moiety as a substrate-binding site. Kinetic analysis of the NADH oxidase activity of EP '-PEG-GluDH quantitatively showed the rate-acceleration effect of the presence of the substratebinding site near the catalytic group [l]. Next, we prepared

Generation of New Enzymes via Covalent Modification of Existing Proteins

“…Additional studies involved the incorporation of 5-thylphenazine (EP + ) into the aforementioned NAD conjugates. Several conjugates were synthesized, including EP + −PEG−GluDH, EP + −GlcDH−NAD + , and EP + −LDH−NAD + . − The EP + −PEG−GluDH conjugate had NADH oxidase activity; the EP + acts as a catalytic group which oxidizes NADH and the glutamate dehydrogenase functions as a substrate-binding site. The schematic catalytic cycle of EP + −PEG−GluDH is illustrated in structure 48-1 .…”

“…EP + −GlcDH−NAD + and EP + −LDH−NAD + conjugates were prepared by attaching both PEG−NAD and PEG−EP + to glucose dehydrogenase and lactate dehydrogenase, respectively. Structures 48-2 and 48 − 3 show the catalytic cycle of these oxidases (EP + −GlcDH−NAD + and EP + −LDH−NAD + , respectively). , The catalytic cycle of EP + −GlcDH−NAD + starts with the reduction of the NAD + moiety. Then the NADH moiety is reoxidized by the ethylphenazine (EP + ) group by oxidation of glucose.…”

“…Kinetic analysis of the EP + −GlcDH−NAD + -catalyzed reaction shows that this mechanism involving high effective concentration, coupling of successive catalytic reactions, and multiple connections between the two kinds of catalytic sites mimics the catalytic cycles of natural enzymes. EP + −LDH−NAD + shows a different catalytic cycle (structure 48-3 ) from that of EP + −GlcDH−NAD + . There are two more enzyme forms containing an NAD + or NADH moiety in the coenzyme-binding site of the conjugate.…”

“…Kinetic analysis of the NADH oxidase activity of EP + -PEG-GluDH quantitatively shows the effect of the presence of the substrate-binding site near the catalytic group. 174 EP + -GlcDH-NAD + and EP + -LDH-NAD + conjugates were prepared by attaching both PEG-NAD and PEG-EP + to glucose dehydrogenase and lactate dehydrogenase, respectively. Structures 48-2 and 48-3 show the catalytic cycle of these oxidases (EP + -GlcDH-NAD + and EP + -LDH-NAD + , respectively).…”

“…Structures 48-2 and 48-3 show the catalytic cycle of these oxidases (EP + -GlcDH-NAD + and EP + -LDH-NAD + , respectively). 173,174 The catalytic cycle of EP + -GlcDH-NAD + starts with the reduction of the NAD + moiety. Then the NADH moiety is reoxidized by the ethylphenazine (EP + ) group by oxidation of glucose.…”