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 dissocation 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 (rotenone, antimycin A, o-phenanthroline, etc.) which inhibited the electon transport in energy-linked electron transport system and some flavoproteins.
Sulfhydryl groups seem to be involved in catalytic cycle by the relatively insensitive to the p-chloromercurylphenyl sulfonate, N-ethylmaleimide, iodoactamide, nor in binding of flavin and reduced nicotineamide adenine dinucleotide, in contrast to NADH dehydrogenase in different system.
Tyrosine group may be involved in binding of flavin to the apoenzyme because of the extreme sensitivity of apoenzyme to the $I_2$ in KI, but relative insensitivity of holoenzyme. Serine group and histidine, tryptophan groups are not involved in catalytic cycle by the experments of diisopropylfluorophosphate and photooxidation in methylene blue, respectively.
A possible role for this enzyme in coupling to a mixed function oxidase, which it was known to be supplied with electrons, is envisaged.
Phenol 에서 자란 P. putida 에서 NADH:(acceptor) oxidoreductase (EC 1.1.99-) 를 약 30 배 정도로 분리하였다. 이 효소에 대해 생체내에서의 전자 받는 것이 어느 것인지는 아직 알려지지 않았지만, hydroxylation 계에서 관여되리라 추측이 된다.
이 효소의 조효소는 flavin 계로서, 이것의 분리 상수가 상당히 커서 분리하는 동안 떨어져나와 apoenzyme 을 만든다.
이 효소는 cytochrome C 와 DCIP 를 환원시킬 수 있었는데 DCIP가 더 전자를 잘 받았다. 그리고 NADH 가 상대적으로 더 NADP 보다 specific 하였다. NADH 와 DCIP 의 이 효소에 대한 Km 값은 1.4×$10^{-5}M$ 과 6×$10^{-5}M$ 이고 이 효소의 FMN 에 대한 $K_D$ 는 1.5×$10^{-7}M$ 이고 FAD 에 대해서는 2.1×$10^{-6} M$ 이다. FMN 이 이 효소의 조효소로 추측이 되어진다. 또한 금속이온이 효소활성에 관여되지 않은 것 같다.
이 효소는 kinetics 적으로 ping-pong mechanism 을 나타내는데 이 효소가 산화된 형태와 환원된 형태가 존재한다는 것을 알 수가 있다. 여기에는 -SH group 이 관여하지 않는다. 또한 FMN 과 NADH 의 결합에도 관여되지 않는다. Tyrosine 이 FMN 결합에 관여할 것 같다. Histidine, tryptophan, serine group이 반응에 관여않는데, 어떤 group 이 이러한 산화 환원 반응에 관여하는지는 더 연구할 과제이다.
