Environments and mechanistic roles of the tyrosine residues of Δ5-3-ketosteroid isomerase

Yaw Kuen Li, Athan Kuliopulos, Albert S. Mildvan, Paul Talalay

Research output: Contribution to journalArticle

Abstract

Δ5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni converts Δ5-3-ketosteroids to Δ4-3-ketosteroids by a stereoselective and conservative transfer of the 4β-proton to the 6β-position. The 109.5-fold enzymatic rate acceleration can be attributed to a concerted rate-limiting enolization in which Tyr-14 and Asp-38, positioned orthogonally, act as general acid and base, respectively. The pKa value of the phenolic hydroxyl group of Tyr-14 of the Y55F/Y88F double mutant is 11.6 ± 0.2 by UV titration. However, the fluorescence titration of Tyr-14 shows biphasic sigmoidal behavior with apparent pKa values of 9.5 and 11.5. This suggests the assistance of a basic residue at the active site, possibly a lysine or tyrosine residue. Mutations of each of the four lysine residues K119L, K108Q, K92Q, and K60Q lowered specific activities only slightly to between 43% and 98% of the wild-type enzyme. Similarly, mutations of Tyr-55, Tyr-88, or both to phenylalanine led to only 2-4-fold reductions in catalytic activity. These findings suggest that despite the enormous difference between the pKa value of Tyr-14 (11.6) and that of the 3-carbonyl group of the steroid (ca. pKa -7), the reaction may rely on the concerted participation of Tyr-14 and Asp-38 only. The apparent pKa value of 9.5 in the fluorescence titration of Tyr-14 and in kinetic measurements probably results from conformational changes of the enzyme. The unusually high pKa value of Tyr- 14 of 11.6 ± 0.2 was used to estimate a local dielectric constant of 18 ± 2 near this residue. UV absorption spectra of each of the three tyrosine double and single mutants reveal that the molar absorbances of Tyr-14 and Tyr-55 are 25-30% greater than those of Tyr-88 or of N-acetyltyrosine amide in solution and are red-shifted by 4-5 nm. Fluorescence studies show that the relative molar fluorescence intensity of Tyr-14 is unusually high (3.9) and that of Tyr-88 is much lower (0.42) in comparison to that of free N-acetyltyrosine amide. Consistent with the estimated dielectric constant of 18 ± 2 near Tyr-14, both the UV absorption spectrum and the fluorescence enhancement of Tyr-14 are mimicked by N-acetyltyrosine amide in 2-propanol, a solvent with a dielectric constant of 18.6. The fluorescences of Tyr-14 and Tyr-55 are strongly mutually quenched, whereas Tyr-88 has no effect on the fluorescence of these residues, and vice versa. The binding of 19-nortestosterone to the enzyme quenches nearly all of the fluorescence of Tyr-14 and Tyr-55, but only partially quenches the fluorescence of Tyr-88. These findings are consistent with the partial X-ray structure of isomerase which reveals that Tyr-14 and Tyr-55 are located in the hydrophobic active site while Tyr-88 is more distant and at least partially exposed to the medium.

Original languageEnglish (US)
Pages (from-to)1816-1824
Number of pages9
JournalBiochemistry®
Volume32
Issue number7
StatePublished - 1993

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steroid delta-isomerase
Tyrosine
Fluorescence
Titration
Amides
Ketosteroids
Permittivity
Lysine
Absorption spectra
Catalytic Domain
Enzymes
Comamonas testosteroni
Nandrolone
Isomerases
Mutation
2-Propanol
Phenylalanine
Hydroxyl Radical
Protons

ASJC Scopus subject areas

  • Biochemistry

Cite this

Li, Y. K., Kuliopulos, A., Mildvan, A. S., & Talalay, P. (1993). Environments and mechanistic roles of the tyrosine residues of Δ5-3-ketosteroid isomerase. Biochemistry®, 32(7), 1816-1824.

Environments and mechanistic roles of the tyrosine residues of Δ5-3-ketosteroid isomerase. / Li, Yaw Kuen; Kuliopulos, Athan; Mildvan, Albert S.; Talalay, Paul.

In: Biochemistry®, Vol. 32, No. 7, 1993, p. 1816-1824.

Research output: Contribution to journalArticle

Li, YK, Kuliopulos, A, Mildvan, AS & Talalay, P 1993, 'Environments and mechanistic roles of the tyrosine residues of Δ5-3-ketosteroid isomerase', Biochemistry®, vol. 32, no. 7, pp. 1816-1824.
Li, Yaw Kuen ; Kuliopulos, Athan ; Mildvan, Albert S. ; Talalay, Paul. / Environments and mechanistic roles of the tyrosine residues of Δ5-3-ketosteroid isomerase. In: Biochemistry®. 1993 ; Vol. 32, No. 7. pp. 1816-1824.
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abstract = "Δ5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni converts Δ5-3-ketosteroids to Δ4-3-ketosteroids by a stereoselective and conservative transfer of the 4β-proton to the 6β-position. The 109.5-fold enzymatic rate acceleration can be attributed to a concerted rate-limiting enolization in which Tyr-14 and Asp-38, positioned orthogonally, act as general acid and base, respectively. The pKa value of the phenolic hydroxyl group of Tyr-14 of the Y55F/Y88F double mutant is 11.6 ± 0.2 by UV titration. However, the fluorescence titration of Tyr-14 shows biphasic sigmoidal behavior with apparent pKa values of 9.5 and 11.5. This suggests the assistance of a basic residue at the active site, possibly a lysine or tyrosine residue. Mutations of each of the four lysine residues K119L, K108Q, K92Q, and K60Q lowered specific activities only slightly to between 43{\%} and 98{\%} of the wild-type enzyme. Similarly, mutations of Tyr-55, Tyr-88, or both to phenylalanine led to only 2-4-fold reductions in catalytic activity. These findings suggest that despite the enormous difference between the pKa value of Tyr-14 (11.6) and that of the 3-carbonyl group of the steroid (ca. pKa -7), the reaction may rely on the concerted participation of Tyr-14 and Asp-38 only. The apparent pKa value of 9.5 in the fluorescence titration of Tyr-14 and in kinetic measurements probably results from conformational changes of the enzyme. The unusually high pKa value of Tyr- 14 of 11.6 ± 0.2 was used to estimate a local dielectric constant of 18 ± 2 near this residue. UV absorption spectra of each of the three tyrosine double and single mutants reveal that the molar absorbances of Tyr-14 and Tyr-55 are 25-30{\%} greater than those of Tyr-88 or of N-acetyltyrosine amide in solution and are red-shifted by 4-5 nm. Fluorescence studies show that the relative molar fluorescence intensity of Tyr-14 is unusually high (3.9) and that of Tyr-88 is much lower (0.42) in comparison to that of free N-acetyltyrosine amide. Consistent with the estimated dielectric constant of 18 ± 2 near Tyr-14, both the UV absorption spectrum and the fluorescence enhancement of Tyr-14 are mimicked by N-acetyltyrosine amide in 2-propanol, a solvent with a dielectric constant of 18.6. The fluorescences of Tyr-14 and Tyr-55 are strongly mutually quenched, whereas Tyr-88 has no effect on the fluorescence of these residues, and vice versa. The binding of 19-nortestosterone to the enzyme quenches nearly all of the fluorescence of Tyr-14 and Tyr-55, but only partially quenches the fluorescence of Tyr-88. These findings are consistent with the partial X-ray structure of isomerase which reveals that Tyr-14 and Tyr-55 are located in the hydrophobic active site while Tyr-88 is more distant and at least partially exposed to the medium.",
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TY - JOUR

T1 - Environments and mechanistic roles of the tyrosine residues of Δ5-3-ketosteroid isomerase

AU - Li, Yaw Kuen

AU - Kuliopulos, Athan

AU - Mildvan, Albert S.

AU - Talalay, Paul

PY - 1993

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N2 - Δ5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni converts Δ5-3-ketosteroids to Δ4-3-ketosteroids by a stereoselective and conservative transfer of the 4β-proton to the 6β-position. The 109.5-fold enzymatic rate acceleration can be attributed to a concerted rate-limiting enolization in which Tyr-14 and Asp-38, positioned orthogonally, act as general acid and base, respectively. The pKa value of the phenolic hydroxyl group of Tyr-14 of the Y55F/Y88F double mutant is 11.6 ± 0.2 by UV titration. However, the fluorescence titration of Tyr-14 shows biphasic sigmoidal behavior with apparent pKa values of 9.5 and 11.5. This suggests the assistance of a basic residue at the active site, possibly a lysine or tyrosine residue. Mutations of each of the four lysine residues K119L, K108Q, K92Q, and K60Q lowered specific activities only slightly to between 43% and 98% of the wild-type enzyme. Similarly, mutations of Tyr-55, Tyr-88, or both to phenylalanine led to only 2-4-fold reductions in catalytic activity. These findings suggest that despite the enormous difference between the pKa value of Tyr-14 (11.6) and that of the 3-carbonyl group of the steroid (ca. pKa -7), the reaction may rely on the concerted participation of Tyr-14 and Asp-38 only. The apparent pKa value of 9.5 in the fluorescence titration of Tyr-14 and in kinetic measurements probably results from conformational changes of the enzyme. The unusually high pKa value of Tyr- 14 of 11.6 ± 0.2 was used to estimate a local dielectric constant of 18 ± 2 near this residue. UV absorption spectra of each of the three tyrosine double and single mutants reveal that the molar absorbances of Tyr-14 and Tyr-55 are 25-30% greater than those of Tyr-88 or of N-acetyltyrosine amide in solution and are red-shifted by 4-5 nm. Fluorescence studies show that the relative molar fluorescence intensity of Tyr-14 is unusually high (3.9) and that of Tyr-88 is much lower (0.42) in comparison to that of free N-acetyltyrosine amide. Consistent with the estimated dielectric constant of 18 ± 2 near Tyr-14, both the UV absorption spectrum and the fluorescence enhancement of Tyr-14 are mimicked by N-acetyltyrosine amide in 2-propanol, a solvent with a dielectric constant of 18.6. The fluorescences of Tyr-14 and Tyr-55 are strongly mutually quenched, whereas Tyr-88 has no effect on the fluorescence of these residues, and vice versa. The binding of 19-nortestosterone to the enzyme quenches nearly all of the fluorescence of Tyr-14 and Tyr-55, but only partially quenches the fluorescence of Tyr-88. These findings are consistent with the partial X-ray structure of isomerase which reveals that Tyr-14 and Tyr-55 are located in the hydrophobic active site while Tyr-88 is more distant and at least partially exposed to the medium.

AB - Δ5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni converts Δ5-3-ketosteroids to Δ4-3-ketosteroids by a stereoselective and conservative transfer of the 4β-proton to the 6β-position. The 109.5-fold enzymatic rate acceleration can be attributed to a concerted rate-limiting enolization in which Tyr-14 and Asp-38, positioned orthogonally, act as general acid and base, respectively. The pKa value of the phenolic hydroxyl group of Tyr-14 of the Y55F/Y88F double mutant is 11.6 ± 0.2 by UV titration. However, the fluorescence titration of Tyr-14 shows biphasic sigmoidal behavior with apparent pKa values of 9.5 and 11.5. This suggests the assistance of a basic residue at the active site, possibly a lysine or tyrosine residue. Mutations of each of the four lysine residues K119L, K108Q, K92Q, and K60Q lowered specific activities only slightly to between 43% and 98% of the wild-type enzyme. Similarly, mutations of Tyr-55, Tyr-88, or both to phenylalanine led to only 2-4-fold reductions in catalytic activity. These findings suggest that despite the enormous difference between the pKa value of Tyr-14 (11.6) and that of the 3-carbonyl group of the steroid (ca. pKa -7), the reaction may rely on the concerted participation of Tyr-14 and Asp-38 only. The apparent pKa value of 9.5 in the fluorescence titration of Tyr-14 and in kinetic measurements probably results from conformational changes of the enzyme. The unusually high pKa value of Tyr- 14 of 11.6 ± 0.2 was used to estimate a local dielectric constant of 18 ± 2 near this residue. UV absorption spectra of each of the three tyrosine double and single mutants reveal that the molar absorbances of Tyr-14 and Tyr-55 are 25-30% greater than those of Tyr-88 or of N-acetyltyrosine amide in solution and are red-shifted by 4-5 nm. Fluorescence studies show that the relative molar fluorescence intensity of Tyr-14 is unusually high (3.9) and that of Tyr-88 is much lower (0.42) in comparison to that of free N-acetyltyrosine amide. Consistent with the estimated dielectric constant of 18 ± 2 near Tyr-14, both the UV absorption spectrum and the fluorescence enhancement of Tyr-14 are mimicked by N-acetyltyrosine amide in 2-propanol, a solvent with a dielectric constant of 18.6. The fluorescences of Tyr-14 and Tyr-55 are strongly mutually quenched, whereas Tyr-88 has no effect on the fluorescence of these residues, and vice versa. The binding of 19-nortestosterone to the enzyme quenches nearly all of the fluorescence of Tyr-14 and Tyr-55, but only partially quenches the fluorescence of Tyr-88. These findings are consistent with the partial X-ray structure of isomerase which reveals that Tyr-14 and Tyr-55 are located in the hydrophobic active site while Tyr-88 is more distant and at least partially exposed to the medium.

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