CRYSTAL-STRUCTURE AND ENZYME MECHANISM OF DELTA(5)-3-KETOSTEROID ISOMERASE FROM PSEUDOMONAS-TESTOSTERONI

Citation
Hs. Cho et al., CRYSTAL-STRUCTURE AND ENZYME MECHANISM OF DELTA(5)-3-KETOSTEROID ISOMERASE FROM PSEUDOMONAS-TESTOSTERONI, Biochemistry, 37(23), 1998, pp. 8325-8330
Citations number
30
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
37
Issue
23
Year of publication
1998
Pages
8325 - 8330
Database
ISI
SICI code
0006-2960(1998)37:23<8325:CAEMOD>2.0.ZU;2-Y
Abstract
Bacterial Delta(5)-3-ketosteroid isomerase (KSI) from Pseudomonas test osteroni has been intensively studied as a prototype for understanding an enzyme-catalyzed allylic rearrangement involving intramolecular pr oton 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 carboxy l 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 catalyti c 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 stru cture of this enzyme at 2.3 Angstrom resolution. The crystal structure revealed that the active site environment of P. testosteroni KSI is n early identical to that of Pseudomonas putida KSI, whose structure in complex with a reaction intermediate analogue we have determined recen tly. Comparison of the two structures clearly indicates that the two K SIs 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) CO OH. 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.