Crystal structure and enzyme mechanism of Delta(5)-3-ketosteroid isomerase from Pseudomonas testosteroni

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Bacterial Delta(5)-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni has been intensively studied as a prototype for understanding an enzyme-catalyzed allylic rearrangement involving intramolecular proton transfer. Asp(38) serves as a general base to abstract the proton from the steroid C4-H, which is a much stronger base than the carboxyl group of this residue. This unfavorable proton transfer requires 11 kcal/mol of energy which has to be provided by favorable interactions between catalytic residues and substrate in the course of the catalytic reaction. How this energy is provided at the active site of KSI has been a controversial issue, and inevitably the enzyme mechanism is not settled. To resolve these issues, we have determined the crystal structure of this enzyme at 2.3 Angstrom resolution. The crystal structure revealed that the active site environment of P. testosteroni KSI is nearly identical to that of Pseudomonas putida KSI, whose structure in complex with a reaction intermediate analogue we have determined recently. Comparison of the two structures clearly indicates that the two KSIs should share the same enzyme mechanism involving the stabilization of the dienolate intermediate by the two direct hydrogen bonds to the dienolate oxyanion, one from Tyr(14) OH and the other from Asp(99) COOH. Mutational analysis of the two residues and other biochemical data strongly suggest that the hydrogen bond of Tyr(14) provides the more significant contribution than that of Asp(99) to the requisite 11 kcal/mol of energy for the catalytic power of KSI.
Publisher
AMER CHEMICAL SOC
Issue Date
1998-06
Language
English
Article Type
Article
Keywords

PUTIDA BIOTYPE-B; DELTA-5-3-KETOSTEROID ISOMERASE; PROTON-TRANSFER; CATALYTIC MECHANISM; TRANSITION-STATES; CARBON ACIDS; ABSTRACTION; RESIDUES; MUTANTS; D38N

Citation

BIOCHEMISTRY, v.37, no.23, pp.8325 - 8330

ISSN
0006-2960
DOI
10.1021/bi9801614
URI
http://hdl.handle.net/10203/77658
Appears in Collection
BS-Journal Papers(저널논문)
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