NADH dehydrogenase from phehol-grown pseudomonas putida = 페놀에서 자란 Pseudomonas putida 의 NADH dehydrogenase 에 관한 연구

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NADH dehydrogenase (EC 1.6.99-) has been known to be related with many aromatic compound hydroxylation system in $\underline{Pseudomonas}$ species. But phenol hydroxylase in $\underline{Pseudomonas}$ species has been failed to be detected in cell-free extract. In this, 30-fold induced NADH dehydrogenase in phenol minimal medium compared with the glucose or succinate medium was partially purified 30-fold by ammonium sulfate fractionation, Bio-Gel A-0.5 chromatography, DEAE-cellulose chromatography in 16% yield, and studied the general characterization and some mechanism. This enzyme could reduce the cytochrome c, dichlorophenolindophenol, which is 1- and 2- electron acceptor, respectively, relatively specific to NADH. By steady state kinetic study, this enzyme showed a so called ``ping-pong mechanism``, in which Michaelis constants, $K_m$, for 2, 6-dichlorophenolindophenol and reduced nicotineamide adenine dinucleotide are 1.4×$10^{-4}$ M and 6.0×$10^{-5}$ M, respectively, and the maxium velocity at infinite concentrations of both reactants is 0.4 Δμmole/$\min$ as measured the reduction of 2, 6-dichlorophenolindophenol at 600 nm using the millimolar absorbance coefficient, 21.0 $mM^{-1}cm^{-1}$ at 0.05M potassium phosphate buffer, pH 7.6, 25℃. During the purification, the enzyme activity was lost because bound flavin(s) was dissociated from the holoenzyme. By incubation of apoenzyme with flavin, the dissociation constants, $K_D$, for the ``apoenzyme-flavin mononucleotide complex`` and ``apoenzyme-flavin adenine dinucleotide complex`` are 1.5×$10^{-7}$M and 2.1×$10^{-6}$M, respectively, which are 1000 times higher than those of other flavoproteins. There is no additative metal ion effect on the enzyme activity such $Fe^{+3}$, $Cu^{+2}$, $Ca^{+2}$, etc., and relatively insensitive to various metal-chelating agents such as EDTA, α,α`` -dipyridyl etc. So it may not be related with metal ions in the enzyme activity. Also, it is insensitive to the inhibitors...
Lee, Hyun-Jae이현재
한국과학기술원 : 생물공학과,
Issue Date
62410/325007 / 000771093

학위논문 (석사) - 한국과학기술원 : 생물공학과, 1979.2, [ iv, 74 p. ]

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