Properties of purified L-ornithine decarboxylase (EC 4.1.1.17) from Tetrahymena thermophila.
Ornithine decarboxylase (ornithine carboxy lyase; EC 4.1.1.17) (ODC) from Tetrahymena thermophila was purified 6,300 fold employing fractionated ammonium sulfate precipitation, gel permeation chromatography on Sephadex G-150, ion exchange chromatography on DEAE-Sepharose CL-6B, and preparative isoelectric focussing. The product obtained in 24% yield was a preparation of the specific activity of 10,200 nmol CO2.h-1.mg-1. The purified enzyme was rather stable at 37 degrees C (14% loss of activity within 1 h). The molecular and catalytic properties of this enzyme were investigated. The isoelectric point was 5.7 and the molecular weight (MW) was estimated to be 68,000 under nondenaturing conditions. The pH optimum was between 6.0 and 7.0, the Km for the substrate L-ornithine was 0.11 mM, and the Km for the cofactor pyridoxal 5-phosphate was 0.12 microM; the product of ODC catalysis, putrescine, was a poor inhibitor with an estimated Ki of about 10 mM. The enzyme was inhibited competitively by D-ornithine with a Ki of 1.6 mM and by alpha-difluoromethylornithine with a Ki of 0.15 mM. The latter one, an enzyme activated irreversible inhibitor of mammalian ODC, inactivated the enzyme from T. thermophila at high concentrations with a half life time of 14 min. Other basic amino acids, e.g. L-lysine, L-arginine, and L-histidine, were neither substrates nor inhibitors of the enzyme, as were the diamines 1,3-diaminopropanol and cadaverine, the polyamines spermidine and spermine and the cosubstrate analogues pyridoxal and pyridoxamine-5-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)
Eichler
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Institut für Biochemie, Universität Heidelberg, Federal Republic of Germany.
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008970:Molecular Weight; D009955:Ornithine Decarboxylase / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013768:Tetrahymena / Q000201:enzymology*
D009955:Ornithine Decarboxylase