Purification and Mechanism of Action of a Steroid -5ß-Dehydrogenase

From the ¹ From the Ben May Laboratory for Cancer Research, The University of Chicago, Chicago, Illinois 60637, and the Department of Pharmacology and Experimental Therapeutics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

A soluble ⁴-5ß-dehydrogenase has been isolated from extracts of Pseudomonas testosteroni grown on a steroid-containing medium. This enzyme introduces the ⁴-double bond into C₁₉ and C₂₁ steroids in which the A:B ring fusion has the cis configuration. The enzyme has been purified about 100-fold from acetone powders of the microorganism by a procedure involving precipitation of nucleic acids with protamine; fractionation with ammonium sulfate; chromatography on diethylaminoethyl-cellulose; and calcium phosphate gel adsorption. The purified enzyme oxidized about 6.5 µmoles of 5ß-androstane-3,17-dione per min per mg of protein.

Phenazine methosulfate is an efficient artificial acceptor in the ⁴-5ß-dehydrogenase reaction. Various quinones, notably 1,2- and 1,4-naphthoquinone, 1,4-benzoquinone, menadione, and coenzyme Q₁₀, were able to replace phenazine methosulfate in the reaction, but other quinones and acceptors of other chemical types were inactive.

A simple aerobic assay for steroid -dehydrogenase activity is described. It depends upon measurement of the nonenzymatic reduction of cytochrome c (at 550 mµ) by the reduced phenazine methosulfate formed in the enzymatic oxidation. An interesting blank reaction occurs between phenazine methosulfate and cytochrome c in the absence of enzyme or steroid. The rate of this reaction is markedly accelerated by light and at high pH. The reaction rate is proportional to the concentration of phenazine methosulfate, but independent of that of cytochrome c. These findings suggest that a product of the light-catalyzed dismutative decomposition of the dye is the reductant of cytochrome c.

Treatment with acid ammonium sulfate inactivated the enzyme almost completely, and a substantial portion of the original activity could be restored by addition of a low concentration (K_m = 0.33 µm) of flavin mononucleotide but not by flavin adenine dinucleotide.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				P. Talalay A Fascination with Enzymes: The Journey Not the Arrival Matters J. Biol. Chem., August 12, 2005; 280(32): 28829 - 28847. [Full Text] [PDF]