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3,4-Dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione 4,5-Dioxygenase from Nocardia restrictus

I. ISOLATION OF THE ENZYME AND STUDY OF ITS PHYSICAL AND CHEMICAL PROPERTIES

From the ¹ From the School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706

3,4-Dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione-4,5-dioxygenase was purified to a state of apparent homogeneity from Nocardia restrictus. The molecular weight of the enzyme was estimated to be around 280,000 from sedimentation equilibrium and sedimentation velocitydiffusion data. The physicochemical properties described include: sedimentation coefficient (s⁰_20,w = 10.42 S), diffusion coefficient (D⁰_20,w = 2.55 x 10^-7 cm² sec^-1), frictional ratio (f/f₀ = 1.47), and apparent partial specific volume (v = 0.733 ml g^-1). The enzyme has an ultraviolet absorption maximum at 280 mµ, and the specific absorbance (E^0.1%_{1 cm}) is 0.93. Quantitative analytical data show 1.13 g-atoms of ferrous iron and 9 half-cystinyl residues per mole of the enzyme.

Metal-chelating agents, including o-phenanthroline, 8-hydroxyquinoline, and ,'-dipyridyl, inhibited the enzyme, and the inhibition was noncompetitive with respect to both organic substrate and molecular oxygen. Sulfhydryl inhibitors also inhibited the enzyme at the concentration of 1 mm.

Substrate specificity studies indicated that substitution of an alkyl group at a position adjacent to the dihydroxyl group was required for maximal activity. Introduction of a bulky alkyl group at other positions rendered the substrate less reactive.

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