5-Androstene-3,17-dione isomerization

Figure 4-12 The mechanism for the isomerization of A5 — A4-androstene-3,17-dione with optimized geometries for the seven key-structures.

Fig. 3. Various types of reaction catalyzed by GST. Besides the formation of thioethers, GST catalyze thiolysis, denitrozation, isomerization, and peroxidase-type reactions (see text). As examples of such reactions, the metabolism of p-nitrophenyl acetate, nitroglycerine, A -androstene-3,17-dione, and cumene hydroperoxide are shown in a, b, c, and d, respectively.

The distribution of 0 between the hydroxyl and hydroxymethylene functions of 19a and 19b, determined by the fragmentation pattern of these two species during mass spectrometry, could only result from addition of the carboxyl of the enzyme to the a face of the steroid. Had the carboxyl added to the spiro carbon from the jS face, the 0 would have been located in the hydroxymethylene function of 19a. Clearly, if the binding of the oxiranyl steroids is analogous to that of steroid substrates, Asp-38 could not be involved in the normal intramolecular proton transfer associated with the isomerization reaction. However, as recently emphasized by Pollack ei al., the oxiranyl steroids could conceivably bind to the active site in an upsidedown orientation in comparison to that of the substrate steroids (133). In this case, Asp-38 would still be the most probable base involved in the intramolecular proton transfer reaction. Perhaps upsidedown binding accounts for the report that the C-4a hydrogen of 5-androstene-3,17-dione undergoes slow labilization in the presence of the isomerase (134, 135). 

Androstene-3,17-dione is isomerized to 4-androstene-3,17-dione by hydroxide ion. Propose a mechanism for this reaction. 

Sites of Reaction. la-Hydroxylation. Dodson, Goldkamp, and Muir (D-162, D-163) obseryed la-hydxoxyla.iioxiVii Penicillium sp. on 4-androstene-3,17-dione, 5o -androstane-3,17-dione, and 3j3-hydroxy-5-androsten-17-one (dehydroepiandro-sterone). The organism also oxidizes the 3-hydroxyl and isomerizes the double bond. 

A separation in the metabolic route occurs on the reduction of the double bond of A -androstene-3,17-dione (LV), affording two saturated diketones isomeric at Cs. These are rings A/B trans, androstane-3,17-dione (LX), and rings A/B cis, etiocholane-3,17-dione (LXI). Both of these diketones have been isolated from urine. " That these two diketones are true metabolites of testosterone has been shown by the isolation of the tagged substances after injection of deuterium-labeled hormone. Once the metabolic course has diverged upon the reduction of the a, unsaturated ketone to the isomeric diketones, there is no crossing over from one branch of the pathway to the other. Thus, after the administration of either deuterium-labeled or nonisotopic androstane-3,17-dione (LX), only... 

In one of the earliest studies [39], we carried out the purification of A5 4 3-oxosteroid isomerase from a crude extract of Pseudomonas testosteroni by affinity pardoning in a poly(ethylene glycol)/dextran system. The macroligand was prepared by covalent coupling on poly(ethylene glycol) (PEG) of the same steroid derivative as previously described in Fig. 14.2. The resulting macroligand PEG-E,i had an inhibition constant Ki around 5 pM towards the isomerization of A5-androstene-3,17-dione, by isomerase. 

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