ULTRAVIOLET RESONANCE RAMAN-SPECTROSCOPY OF DELTA(5)-3-KETOSTEROID ISOMERASE REVISITED - SUBSTRATE POLARIZATION BY ACTIVE-SITE RESIDUES

The Delta(5)-3-ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testo steroni promotes extremely rapid conversion of Delta(5)- to Delta(4)-3 -ketosteroids by a conservative intramolecular proton transfer via an enolic intermediate. The competitive inhibitor 19-nortestosterone disp lays marked spectroscopic changes upon binding to the enzyme, but the mechanisms responsible for these changes have not been unequivocally e stablished, Ultraviolet resonance Raman (UVRR) spectra are reported fo r 19-nortestosterone in acid solutions and for this ligand when bound to Delta(5)-3-ketosteroid isomerase, as well as to its D38N and Y14F/D 38N mutants, The frequencies of UVRR bands associated with C=O and C=C stretching can be used to monitor the state of polarization of the en one fragment of the steroid and the effects of the catalytic side chai ns, Tyr-14 and Asp-38, on these polarizations. Strong polarization is indicated by marked frequency downshifts of the C=O and C=C bands in t he native protein; the downshifts are diminished by the mutations of t hese catalytic residues. The lower polarizing effects of the Y14F and D38N single mutants and the Y14F/D38N double mutant indicate that most of the polarization of the conjugated ketone is attributable to hydro gen-bond donation by the hydroxyl group of Tyr-14. A smaller contribut ion of Asp-38 is detected which is, in part, cooperative with that of Tyr-14. Reference spectra of hydrogen-bonded and protonated forms of 1 9-nortestosterone are reassigned, on the basis of the species identifi cation of D. C. Hawkinson and R. M. Pollack [(1993) Biochemistry 32, 6 94-698]. On the basis of this reassignment, the strength of the 19-nor testosterone polarization produced by the native enzyme is intermediat e between complete protonation and the hydrogen-bonding environment of 10 M hydrochloric acid. Since the UVRR spectrum of Tyr-14 is unpertur bed upon binding of the steroid, the hydrogen bond to the carbonyl gro up of 19-nortestosterone may be compensated by a second hydrogen bond to Tyr-l4 from another donor, possibly a backbone NH or a bound water molecule, This compensating hydrogen bond could lower the free energy of the enzyme transition state, in which the hydroxyl proton of Tyr-14 helps to dissipate the negative charge that accumulates on the steroi d carbonyl group.