Alicyclic acid metabolism in plants 10. Partial purification and some properties of 3-dehydroquinate synthase from Phaseolus mungo seedlings

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Dehydroquinate synthase from Phaseolus mungo seedlings was purified 120-fold by DE-23, hydroxylapatite and Sephadex G-100 column chromatography. The final preparation was free of dehydroquinate hydro-lyase and NAD(P)H_2 oxidase. The dehydroquinate synthase required Co^<2+> and NAD as cofactors. Co^<2+> could be replaced by Cu^<2+> at 0.1 mM, but Cu^<2+> at higher levels was inhibitory. None of the other metal ions tested activated the enzyme. Some activity was observed in the absence of added Co^<2+> and this activity was inhibited by EDTA but not by diethyldithiocarbamate, NaN_3 or NaCN. Heavy metal ions, such as Ag^+ and Hg^<2+>, and p-chloromercuribenzoate strongly inhibited the enzyme activity. Of the pyridine nucleotides tested only NAD was required for the maximum activity of the enzyme. In the absence of NAD, the enzyme retained 30 to 40% of the activity obtained with added NAD. The apparent Km value for DAHP at pH 7.4 was about 23 μM. The enzyme activity appeared to be maximum at about pH 8.5. However, the characteristics of the enzyme were studied at pH 7.4, because of the lability of the enzyme under alkaline conditions. Arrhenius plot of the enzyme reaction showed a break at about 21℃, and below this critical temperature the activation energy increased.
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