High-resolution crystal structures of Δ5-3-ketosteroid isomerase with and without a reaction intermediate analogue

Suhng Wook Kim, Sun Shin Cha, Hyun Soo Cho, Jeong Sun Kim, Nam Chul Ha, Moon Ju Cho, Soyoung Joo, Kyeong Kyu Kim, Kwan Yong Choi, Byung Ha Oh

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Abstract

Bacterial Δ5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pK(a) values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 Å resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pK(a) appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to contribute significantly to the rate enhancement based on mutational analysis. This observation excludes the residue 101 as a potential catalytic residue and requires that the rate enhancement should be explained solely by Tyr14 and Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14 contributes much more significantly to the rate enhancement than Asp99. Previous studies and the structural analysis strongly suggest that the low- barrier hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength hydrogen bond of Asp99 (~4 kcal/mol), accounts for the required energy of 11 kcal/mol for the transition-state stabilization.

Original languageEnglish
Pages (from-to)14030-14036
Number of pages7
JournalBiochemistry
Volume36
Issue number46
DOIs
Publication statusPublished - 1997 Nov 18

Fingerprint

steroid delta-isomerase
Reaction intermediates
Ketosteroids
Hydrogen
Isomerases
Hydrogen bonds
Pseudomonas putida
Crystal structure
Equilenin
Catalytic Domain
Comamonas testosteroni
Enzymes
Isomerization
Phenylalanine
Structural analysis
Methionine
Stabilization
Steroids
Molecules
Kinetics

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Kim, Suhng Wook ; Cha, Sun Shin ; Cho, Hyun Soo ; Kim, Jeong Sun ; Ha, Nam Chul ; Cho, Moon Ju ; Joo, Soyoung ; Kim, Kyeong Kyu ; Choi, Kwan Yong ; Oh, Byung Ha. / High-resolution crystal structures of Δ5-3-ketosteroid isomerase with and without a reaction intermediate analogue. In: Biochemistry. 1997 ; Vol. 36, No. 46. pp. 14030-14036.
@article{1cee9eec9d604b279ff26f04312ab6be,
title = "High-resolution crystal structures of Δ5-3-ketosteroid isomerase with and without a reaction intermediate analogue",
abstract = "Bacterial Δ5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pK(a) values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 {\AA} resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pK(a) appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to contribute significantly to the rate enhancement based on mutational analysis. This observation excludes the residue 101 as a potential catalytic residue and requires that the rate enhancement should be explained solely by Tyr14 and Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14 contributes much more significantly to the rate enhancement than Asp99. Previous studies and the structural analysis strongly suggest that the low- barrier hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength hydrogen bond of Asp99 (~4 kcal/mol), accounts for the required energy of 11 kcal/mol for the transition-state stabilization.",
author = "Kim, {Suhng Wook} and Cha, {Sun Shin} and Cho, {Hyun Soo} and Kim, {Jeong Sun} and Ha, {Nam Chul} and Cho, {Moon Ju} and Soyoung Joo and Kim, {Kyeong Kyu} and Choi, {Kwan Yong} and Oh, {Byung Ha}",
year = "1997",
month = "11",
day = "18",
doi = "10.1021/bi971546+",
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Kim, SW, Cha, SS, Cho, HS, Kim, JS, Ha, NC, Cho, MJ, Joo, S, Kim, KK, Choi, KY & Oh, BH 1997, 'High-resolution crystal structures of Δ5-3-ketosteroid isomerase with and without a reaction intermediate analogue', Biochemistry, vol. 36, no. 46, pp. 14030-14036. https://doi.org/10.1021/bi971546+

High-resolution crystal structures of Δ5-3-ketosteroid isomerase with and without a reaction intermediate analogue. / Kim, Suhng Wook; Cha, Sun Shin; Cho, Hyun Soo; Kim, Jeong Sun; Ha, Nam Chul; Cho, Moon Ju; Joo, Soyoung; Kim, Kyeong Kyu; Choi, Kwan Yong; Oh, Byung Ha.

In: Biochemistry, Vol. 36, No. 46, 18.11.1997, p. 14030-14036.

Research output: Contribution to journalArticle

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T1 - High-resolution crystal structures of Δ5-3-ketosteroid isomerase with and without a reaction intermediate analogue

AU - Kim, Suhng Wook

AU - Cha, Sun Shin

AU - Cho, Hyun Soo

AU - Kim, Jeong Sun

AU - Ha, Nam Chul

AU - Cho, Moon Ju

AU - Joo, Soyoung

AU - Kim, Kyeong Kyu

AU - Choi, Kwan Yong

AU - Oh, Byung Ha

PY - 1997/11/18

Y1 - 1997/11/18

N2 - Bacterial Δ5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pK(a) values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 Å resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pK(a) appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to contribute significantly to the rate enhancement based on mutational analysis. This observation excludes the residue 101 as a potential catalytic residue and requires that the rate enhancement should be explained solely by Tyr14 and Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14 contributes much more significantly to the rate enhancement than Asp99. Previous studies and the structural analysis strongly suggest that the low- barrier hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength hydrogen bond of Asp99 (~4 kcal/mol), accounts for the required energy of 11 kcal/mol for the transition-state stabilization.

AB - Bacterial Δ5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pK(a) values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 Å resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pK(a) appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to contribute significantly to the rate enhancement based on mutational analysis. This observation excludes the residue 101 as a potential catalytic residue and requires that the rate enhancement should be explained solely by Tyr14 and Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14 contributes much more significantly to the rate enhancement than Asp99. Previous studies and the structural analysis strongly suggest that the low- barrier hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength hydrogen bond of Asp99 (~4 kcal/mol), accounts for the required energy of 11 kcal/mol for the transition-state stabilization.

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