Steroids and steroidases. VI. On the C-17 specificity of the Δ⁵-3-ketoisomerase of Pseudomonas testosteroni and evidence for substrate micelle formation

Abstract

Studies on the acid-, base-, and enzyme-catalyzed isomerizations of several variously C-17 substituted A5-3-ketosteroids have indicated micellar aggregation of relatively non polar substrates in the dilute aqueous methanol assay solvent to be an important consideration when evaluating specificity data. Further evidence that the isomerase is sensitive to the presence of 17a-substituents in its substrates has been obtained and the probability that tyrosine is the acid catalytic group at the active center is discussed.Canadian Journal of Chemistry, 46, 1459 (1968) IntroductionThe literature reports concerning the C-17 specificity of the A5-3-ketoisomerase of Pseudomonas testosteroni (1-3), and, in particular, that cholest-5-en-3-one (16) is not a substrate of the enzyme (I), prompted us to investigate systematically the behavior of a series of variously C-17 substituted As-3-ketosteroids towards acid-, base-, and enzyme-catalyzed isomerizations to the corresponding ap-unsaturated ketones.

Steroids and steroidases. IX. Activation parameters and the mechanism of base- and enzyme-catalyzed isomerizations of androst-5-ene-3,17-dione. The nature of the active center of the Δ5 → Δ4-3-ketosteroid isomerase of Pseudomonas testosteroni

“…4), imidazole itself should be capable of at least partial simulation of the enzymic process by acting a s a bifunctional catalyst.13 That imidazole, alone or mixed with phenol, is in fact (Table IV) not a very effective catalyst would appear to weigh against the Talalay mechanism. We feel that it is more probable that the imidazole group of the active center histidine is in fact the basic group responsible for the removal of the C-4 proton and its subsequent transfer to C-6, and the mechanism we consider to be most consistent with all the present and previous data (1,4,8,14) is shown in Fig. 6.…”

“…Attention was then directed towards further attempts to identify the acidic and basic groups a t the active center. As was recently pointed out (4), the fluorescence (1) and nitration (14) results of Talalay and his co-workers and data on 17-deoxy-substrates (4) make the phenolic group of a tyrosine residue an attractive candidate for the acidic group designated as HA. In order to assist in evaluating the validity of this suggestion, the catalytic effects of various phenols, particularly those of the hydroxypyridine type found to be so effective in catalyzing glucose mutarotation (15), were measured.…”

“…All isomerizations and treatment of data4 were carried out as described previously (4). As before all runs were carried out at least twice and the reproducibility was + 3 % except for the variable temperature enzyme catalyzed reactions.…”

Microbial hydroxylation of steroids. 5. Metabolism of androst-5-ene-3,17-dione and related compounds by Rhizopusarrhizus ATCC 11145

“…The following steroids were prepared by published procedures: Androst-5-ene-3,17-dione (2a) (26), androsta-3,5-diene-17-one (3) (27,28), (5,6a)-epoxy-5a-androstane-3,17-dione (4a) (29), (5,6a)-epoxy-3~-hydroxy-5a-androstan-17-one (46) (I), (5,6~)-epoxy-3~-hydroxy-5~-androstan-l7-one (5a) (I), (433)-epoxy-50-androstane-3,17-dione (6) (16), (4,5a)-epoxy-5a-androstane-3,17-dione (7) (16), and 5-hydroxy-5a-androstane-3,17-dione (8) (1). 3P-Hydroxyandrost-5-ene-17-one (26) was a commercial sample.…”

“…3P-Hydroxyandrost-5-ene-17-one (26) was a commercial sample. 3j3,5,6P-Trihydroxy-5a-androstan-17-one (9a) Perchloric acid (5.5 mL, 6.5% w/v) was added to a refluxing solution of (5,6a)-epoxy-3~-hydroxy-5a-androstan-l7-one (2 g) in acetone (35 mL).…”