High-Resolution Crystal Structures of 5-3-Ketosteroid Isomerase with and without a Reaction Intermediate Analogue
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- 作者：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 ; and Byung-Ha Oh
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：November 18, 1997
- 年：1997
- 卷：36
- 期：46
- 页码：14030 - 14036
- 全文大小：280K
- 年卷期：v.36,no.46(November 18, 1997)
- ISSN：1520-4995
文摘
Bacterial 
5-3-ketosteroid isomerase (KSI) catalyzesa stereospecific isomerization of steroidsubstrates at an extremely fast rate, overcoming a large disparity ofpKa values between a catalyticresidueand its target. The crystal structures of KSI fromPseudomonas putida and of the enzyme in complexwith equilenin, an analogue of the reaction intermediate, have beendetermined at 1.9 and 2.5 Å resolution,respectively. The structures reveal that the side chains ofTyr14 and Asp99 (a newly identifiedcatalyticresidue) form hydrogen bonds directly with the oxyanion of the boundinhibitor in a completely apolarmilieu of the active site. No water molecule is found at theactive site, and the access of bulk solvent isblocked by a layer of apolar residues. Asp99 issurrounded by six apolar residues, and consequently, itspKa appears to be elevated as high as 9.5 to beconsistent with early studies. No interaction wasfoundbetween the bound inhibitor and the residue 101 (phenylalanine inPseudomonas testosteroni andmethioninein P. putida KSI) which was suggested to contributesignificantly to the rate enhancement based onmutational analysis. This observation excludes the residue 101 asa potential catalytic residue and requiresthat the rate enhancement should be explained solely byTyr14 and Asp99. Kinetic analyses of Y14FandD99L mutant enzymes demonstrate that Tyr14 contributes muchmore significantly to the rate enhancementthan Asp99. Previous studies and the structuralanalysis strongly suggest that the low-barrier hydrogenbond of Tyr14 (>7.1 kcal/mol), along with a moderatestrength hydrogen bond of Asp99 (~4kcal/mol),accounts for the required energy of 11 kcal/mol for thetransition-state stabilization.
5-3-ketosteroid isomerase (KSI) catalyzesa stereospecific isomerization of steroidsubstrates at an extremely fast rate, overcoming a large disparity ofpKa values between a catalyticresidueand its target. The crystal structures of KSI fromPseudomonas putida and of the enzyme in complexwith equilenin, an analogue of the reaction intermediate, have beendetermined at 1.9 and 2.5 Å resolution,respectively. The structures reveal that the side chains ofTyr14 and Asp99 (a newly identifiedcatalyticresidue) form hydrogen bonds directly with the oxyanion of the boundinhibitor in a completely apolarmilieu of the active site. No water molecule is found at theactive site, and the access of bulk solvent isblocked by a layer of apolar residues. Asp99 issurrounded by six apolar residues, and consequently, itspKa appears to be elevated as high as 9.5 to beconsistent with early studies. No interaction wasfoundbetween the bound inhibitor and the residue 101 (phenylalanine inPseudomonas testosteroni andmethioninein P. putida KSI) which was suggested to contributesignificantly to the rate enhancement based onmutational analysis. This observation excludes the residue 101 asa potential catalytic residue and requiresthat the rate enhancement should be explained solely byTyr14 and Asp99. Kinetic analyses of Y14FandD99L mutant enzymes demonstrate that Tyr14 contributes muchmore significantly to the rate enhancementthan Asp99. Previous studies and the structuralanalysis strongly suggest that the low-barrier hydrogenbond of Tyr14 (>7.1 kcal/mol), along with a moderatestrength hydrogen bond of Asp99 (~4kcal/mol),accounts for the required energy of 11 kcal/mol for thetransition-state stabilization.