Asp-99 donates a hydrogen bond not to tyr-14 but to the steroid directly in the catalytic mechanism of Delta(5)-3-ketosteroid isomerase from Pseudomonas putida biotype B
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- Title
- Asp-99 donates a hydrogen bond not to tyr-14 but to the steroid directly in the catalytic mechanism of Delta(5)-3-ketosteroid isomerase from Pseudomonas putida biotype B
- Authors
- Choi, G; Ha, NC; Kim, SW; Kim, DH; Park, S; Oh, BH; Choi, KY
- Date Issued
- 2000-02-08
- Publisher
- AMER CHEMICAL SOC
- Abstract
- Delta(5)-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Delta(5)-3-ketosteroids at a rate approaching the diffusion Limit by an intramolecular transfer of a proton. Despite the extensive studies on the catalytic mechanism, it still remains controversial whether the catalytic residue Asp-99 donates a hydrogen bond to the steroid or to Tyr-14. To clarify the role of Asp-99 in the catalysis, two single mutants of D99E and D99L and three double mutants of Y14F/D99E, Y14F/D99N, and Y14F/D99L have been prepared by site-directed mutagenesis, The D99E mutant whose side chain at position 99 is longer by an additional methylene group exhibits nearly the same k(cat) as the wild-type while the D99L mutant exhibits ca. 125-fold lower k(cat) than that of the wild-type. The mutations made at positions 14 and 99 exert synergistic or partially additive effect on k(cat) in the double mutants, which is inconsistent with the mechanism based on the hydrogen-bonded catalytic diad, Asp-99 COOH...Tyr-14 OH ... C3-O of the steroid. The crystal structure of D99E/D38N complexed with equilenin, an intermediate analogue, at 1.9 Angstrom resolution reveals that the distance between Tyr-14 O eta and Glu-99 O epsilon is Ca. 4.2 Angstrom, which is beyond the range for a hydrogen bond, and that the distance between Glu-99 O epsilon and C3-O of the steroid is maintained to be ca, 2.4 Angstrom, short enough for a hydrogen bond to be formed. Taken together, these results strongly support the idea that Asp-99 contributes to the catalysis by donating a hydrogen bond directly to the intermediate.
- Keywords
- 3-OXO-DELTA(5)-STEROID ISOMERASE; DELTA-5-3-KETOSTEROID ISOMERASE; INTERMEDIATE; BINDING; PROTON; MUTANT; D38N
- URI
- https://oasis.postech.ac.kr/handle/2014.oak/20127
- DOI
- 10.1021/bi991579k
- ISSN
- 0006-2960
- Article Type
- Article
- Citation
- BIOCHEMISTRY, vol. 39, no. 5, page. 903 - 909, 2000-02-08
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- There are no files associated with this item.
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