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
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.