1cqs

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{{STRUCTURE_1cqs| PDB=1cqs | SCENE= }}
{{STRUCTURE_1cqs| PDB=1cqs | SCENE= }}
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'''CRYSTAL STRUCTURE OF D103E MUTANT WITH EQUILENINEOF KSI IN PSEUDOMONAS PUTIDA'''
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===CRYSTAL STRUCTURE OF D103E MUTANT WITH EQUILENINEOF KSI IN PSEUDOMONAS PUTIDA===
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==Overview==
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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 kcat as the wild-type while the D99L mutant exhibits ca. 125-fold lower kcat than that of the wild-type. The mutations made at positions 14 and 99 exert synergistic or partially additive effect on kcat in the double mutants, which is inconsistent with the mechanism based on the hydrogen-bonded catalytic dyad, 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 A resolution reveals that the distance between Tyr-14 O eta and Glu-99 O epsilon is ca. 4.2 A, 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 A, 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.
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(as it appears on PubMed at http://www.pubmed.gov), where 10653633 is the PubMed ID number.
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{{ABSTRACT_PUBMED_10653633}}
==About this Structure==
==About this Structure==
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[[Category: Ksi]]
[[Category: Ksi]]
[[Category: Putida lbhb]]
[[Category: Putida lbhb]]
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 13:00:59 2008''
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jun 30 21:09:30 2008''

Revision as of 18:09, 30 June 2008

Image:1cqs.png

Template:STRUCTURE 1cqs

CRYSTAL STRUCTURE OF D103E MUTANT WITH EQUILENINEOF KSI IN PSEUDOMONAS PUTIDA

Template:ABSTRACT PUBMED 10653633

About this Structure

1CQS is a Single protein structure of sequence from Pseudomonas putida. Full crystallographic information is available from OCA.

Reference

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., Choi G, Ha NC, Kim SW, Kim DH, Park S, Oh BH, Choi KY, Biochemistry. 2000 Feb 8;39(5):903-9. PMID:10653633

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