A reinvestigation of the mechanism of Pseudomonas testosteroni .DELTA.5-3-ketosteroid isomerase

Viger, Antoinette; Coustal, S.; Marquet, Andrée

doi:10.1021/ja00392a034

Cited by 36 publications

(28 citation statements)

References 2 publications

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“…It has also been postulated that Asp-38 is the base involved in the proton transfer during the catalytic reaction (Kuliopulos et al, 1987(Kuliopulos et al, , 1989). This interpretation would require that Asp-38 be able to approach the ß side of the steroid ring system as well as the a side, since proton transfer is predominantly 4/3 to 6/3 (Malhotra & Ringhold, 1965;Viger & Marquet, 1977;Viger et al, 1981). A possible solution to this apparent inconsistency would be the existence of free rotation of the steroid about an axis parallel to the plane of the steroid ring system at the active site, thus allowing Asp-38 to have access to both faces of bound steroid.…”

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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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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“…One particularly well-studied enzyme that functions through an enol(ate) intermediate is the 3-oxo-A5-steroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni, which catalyzes the isomerization of 3-oxo-A5-steroids (1) to their conjugated A4-isomers (3) [for reviews, see Pollack et al (1989a) and Schwab and Henderson (1990)]. This enzyme [also called A5-3-ketosteroid isomerase (KSI)1] utilizes the carboxylate of Asp-38 as a base to abstract the 4j9-proton to form a dienol(ate) [2a(b)], with the hydroxyl group of Tyr-14 acting to polarize the carbonyl, either by proton donation or by hydrogen bonding (Benisek et al, 1980;Viger et al, 1981; Bounds & Pollack, 1987;Kuliopulos et al, 1989). Subsequent protonation by the conjugate acid of Asp-38 at C-6jS completes the reaction (Scheme I).…”

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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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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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“…The fact that the stereoselectivity of 2-H abstraction for compound 3a is so low may seem surprising, as enzymes are generally considered to be stereospecific. On the other hand, other steroid-enolizing enzymes known to involve active-site general bases have been shown to be non-stereospecific with respect to proton abstraction (Smith & Brooks, 1977;Viger et al, 1981). The experimental solution n.m.r.…”

Section: Discussionmentioning

confidence: 99%

Conversion of 19-oxo[2β-2H]androgens into oestrogens by human placental aromatase. An unexpected stereochemical outcome

Cole

Robinson

1990

Biochemical Journal

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Aromatase is a cytochrome P-450 enzyme that catalyzes the conversion of androgens into oestrogens via sequential oxidations at the 19-methyl group. Despite intensive investigation, the mechanism of the third step, conversion of the 19-aldehydes into oestrogens, has remained unsolved. We have previously found that a pre-enolized 19-al derivative undergoes smooth aromatization in non-enzymic model studies, but the role of enolization by the enzyme in transformations of 19-oxoandrogens has not been previously investigated. The compounds 19-oxo[2 beta-2H]testosterone and 19-oxo[2 beta-2H]androstenedione have now been synthesized. Exposure of either of these compounds to microsomal aromatase, in the absence of NADPH, for an extended period led to no significant 2H loss or epimerization at C-2, leaving open the importance of an active-site base. However, in the presence of NADPH there was an unexpected substrate-dependent difference in the stereoselectivity of H loss at C-2 in the enzyme-induced aromatization of 19-oxo[2 beta-2H]-testosterone versus 19-oxo[2 beta-2H]androstenedione. The aromatization results for 17 beta-ol derivative 19-oxo[2 beta-2H]-testosterone correspond to about 1.2:1 2 beta-H/2 alpha-H loss from unlabelled 19-oxotestosterone. In contrast, aromatization results for 19-oxo[2 beta-2H]androstenedione correspond to at least 11:1 2 beta-H/2 alpha-H loss from unlabelled 19-oxoandrostenedione. This substrate-dependent stereoselectivity implies a direct role for an enzyme active-site base in 2-H removal. Furthermore, these results argue against the proposal that 2 beta-hydroxylation is the obligatory third step in aromatase action.

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A reinvestigation of the mechanism of Pseudomonas testosteroni .DELTA.5-3-ketosteroid isomerase

Cited by 36 publications

References 2 publications

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

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

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

Conversion of 19-oxo[2β-2H]androgens into oestrogens by human placental aromatase. An unexpected stereochemical outcome

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