An active-site-directed irreversible inhibitor of Δ5-3-ketosteroid isomerase

Pollack, Ralph M.; Kayser, R.; Bevins, Charles L.

doi:10.1016/0006-291x(79)91948-x

Cited by 30 publications

(31 citation statements)

References 15 publications

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“…A binding constant (£¡) for 4 was determined from its inhibition of the enzymatic isomerization of 5-androstene-3,17-dione. For a competitive inhibitor, K¡ can be evaluated by a plot of the reciprocals of first-order rate constants in the presence of various concentrations of inhibitor (0-184 µ ) as a function of inhibitor concentration (Pollack et al, 1979). This plot is linear and gives a K{ of 78 µ for 4.…”

Section: Resultsmentioning

confidence: 99%

“…of the isomerase in the presence of a competitive inhibitor and the subsequent calculation of the binding constant (A") for the inhibitor (Pollack et al, 1979). The reaction cell contained 3.00 mL of phosphate buffer (0.034 M, pH 7.0), 4% (v/v) methanol, 10 µ 5-androstene-3,17-dione as substrate, and varying amounts of the inhibitor 3-amino-l,3,5(10)-estratrien-17/3-oI (0-184 µ ).…”

Section: Methodsmentioning

confidence: 99%

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Nature of the intermediate in the 3-oxo-.DELTA.5-steroid isomerase reaction

Zeng¹,

Bounds²,

Steiner³

et al. 1992

Biochemistry

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The role of Tyr-14 of 3-oxo-delta 5-steroid isomerase (KSI) was probed by analysis of the spectra of 3-amino-1,3,5(10)-estratrien-17 beta-ol (4) and equilenin (5) bound to the active site of KSI. The ultraviolet spectrum of 4 bound to KSI is identical to that for 4 in neutral solution. This observation indicates that Tyr-14 does not protonate the amine group of 4 at the active site. By analogy, it is argued that the 3-oxo group of steroid substrates for KSI is not protonated during the reaction. In contrast, the fluorescence excitation spectra of 5 bound to KSI show characteristics of an ionized phenol, even at pH values as low as 3.8. It is concluded that the pKa of equilenin is perturbed from its value in solution of 9 to less than or equal to 3.5 at the active site of KSI. Similarly, the pKa of the intermediate dienol in the KSI reaction should be lowered to less than or equal to 4.5 when it is bound to KSI. Thus, the function of Tyr-14 as an electrophilic catalyst is likely the stabilization of the anion of the dienol by hydrogen bonding rather than by proton transfer.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Nature of the intermediate in the 3-oxo-.DELTA.5-steroid isomerase reaction

Zeng¹,

Bounds²,

Steiner³

et al. 1992

Biochemistry

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“…Several lines of evidence suggest that steroids may bind to the isomerase in more than one orientation. We have shown that both 3/3-and 17/3-oxiranes inactive the isomerase in an active-site-directed process (Pollack et al, 1979;Bevins et al, 1980). Each forms a covalent bond to Asp-38 (Kayser et al, 1983;, a group known to be at the active site (Martyr & Benisek, 1975;Ogez et al, 1977),…”

Section: Discussionmentioning

confidence: 99%

Orientation, accessibility, and mobility of equilenin bound to the active site of steroid isomerase

Eames

Pollack²,

Steiner³

1989

Biochemistry

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The fluorescent aromatic steroid equilenin, which contains a beta-naphthol moiety, is bound by 3-oxo-delta 5-steroid isomerase. The excitation and emission fluorescence spectra of equilenin when bound to the enzyme, as well as the fluorescence decay time, are indicative of ground-state ionization. In view of the high efficiency of tyrosine quenching, which approaches 100%, the beta-naphthol moiety of equilenin must be in proximity to all three tyrosines of steroid isomerase to account for the observed efficiency of radiationless energy transfer. From the observed response to an external quencher, it appears that enzyme-bound equilenin is largely shielded from solvent. Fluorescence anisotropy measurements indicate a high degree of immobilization of the bound ligand. These models are consistent with proposed models of the enzyme-substrate complex.

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“…In order to compare the reactivity of 2cacyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) with other inhibitors of the bacterial isomerase, I have synthesized pure 3a-spiro-oxiranyl-5a-pregnan-20f-ol (IV) and a 2:1 mixture of the 3#-spiro-oxiranylpregnane (III) + 3a-spiro-oxiranylpregnane (IV). These pregnane derivatives are related to the 3/-spirooxiranyl-5ox-androstan-17#-ol synthesized by Pollack et al (1979). This latter compound is among the most potent irreversible inhibitors of the bacterial enzyme, whereas the opposite stereoisomer, the 3a-spiro-oxiranyl-5a-androstan-1 7,B-ol, was unable to cause enzyme inactivation.…”

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confidence: 99%

“…Since these earlier observations, a number of effective irreversible inhibitors of the bacterial isomerase have been reported. These include the suicide substrates, the 5,1 0-seco-3-oxosteroids synthesized by Batzold & Robinson (1975, the 3f-spiro-oxiranylandrostane derivatives synthesized by Pollack et al (1979) and the aromatase inhibitor 10,B-(1 -oxoprop-2-ynyl)oestr-4-ene-3,17dione (Penning et al, 1981a).…”

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confidence: 99%

Irreversible inhibition of Δ5-3-oxosteroid isomerase by 2-substituted progesterones

Penning¹

1985

Biochemical Journal

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2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) were synthesized and screened as irreversible active-site-directed inhibitors of the delta 5-3-oxosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Both compounds were found to inhibit the purified bacterial enzyme in a time-dependent manner. In either case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond had formed between the inhibitor and the enzyme. Inactivation mediated by compounds (I) and (II) followed pseudo-first-order kinetics, and at higher inhibitor concentrations saturation was observed. The competitive inhibitor 17 beta-oestradiol offered protection against the inactivation mediated by both compounds, and initial-rate studies indicated that compounds (I) and (II) can also act as competitive inhibitors yielding Ki values identical with those generated during inactivation experiments. 2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) thus appear to be active-site-directed. To compare the reactivity of these 2-substituted progesterones with other irreversible inhibitors of the isomerase, 3 beta-spiro-oxiranyl-5 alpha-pregnan-20 beta-ol (III) was synthesized as the C21 analogue of 3 beta-spiro-oxiranyl-5 alpha-androstan-17 beta-ol, which is a potent inactivator of the isomerase [Pollack, Kayser & Bevins (1979) Biochem. Biophys. Res. Commun. 91, 783-790]. Comparison of the bimolecular rate constants for inactivation (k+3/Ki) mediated by compounds (I)-(III) indicated the following order of reactivity: (III) greater than (II) greater than (I). 2-Mercaptoethanol offers complete protection against the inactivation of the isomerase mediated by 2 alpha-cyanoprogesterone (I). Under the conditions of inactivation compound (I) appears to be completely stable, and no evidence could be obtained for enolate ion formation in the presence or absence of enzyme. It is suggested that cyanoprogesterone inactivates the isomerase after direct nucleophilic attack at the electropositive 2-position, and that tautomerization plays no role in the inactivation event. By contrast, 2-mercaptoethanol offers no protection against the inactivation mediated by 2-hydroxymethyleneprogesterone, and under the conditions of inactivation this compound appears to exist in the semi-enolized form.

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An active-site-directed irreversible inhibitor of Δ5-3-ketosteroid isomerase

Cited by 30 publications

References 15 publications

Nature of the intermediate in the 3-oxo-.DELTA.5-steroid isomerase reaction

Nature of the intermediate in the 3-oxo-.DELTA.5-steroid isomerase reaction

Orientation, accessibility, and mobility of equilenin bound to the active site of steroid isomerase

Irreversible inhibition of Δ5-3-oxosteroid isomerase by 2-substituted progesterones

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