Ultraviolet Resonance Raman Spectroscopy of Δ5-3-Ketosteroid Isomerase Revisited: Substrate Polarization by Active-Site Residues

Trace Jordan, Thomas G. Spiro, Janina C. Austin, Qinjian Zhao, Paul Talalay, Albert S. Mildvan

Research output: Contribution to journalArticlepeer-review

Abstract

The Δ5-3-ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni promotes extremely rapid conversion of Δ5- to Δ4-3-ketosteroids by a conservative intramolecular proton transfer via an enolic intermediate. The competitive inhibitor 19-nortestosterone displays marked spectroscopic changes upon binding to the enzyme, but the mechanisms responsible for these changes have not been unequivocally established. Ultraviolet resonance Raman (UVRR) spectra are reported for 19-nortestosterone in acid solutions and for this ligand when bound to Δ5-3-ketosteroid isomerase, as well as to its D38N and Y14F/D38N 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 enone fragment of the steroid and the effects of the catalytic side chains, 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 the native protein; the downshifts are diminished by the mutations of these catalytic residues. The lower polarizing effects of the YMF and D38N single mutants and the Y14F/D38N double mutant indicate that most of the polarization of the conjugated ketone is attributable to hydrogen-bond donation by the hydroxyl group of Tyr-14. A smaller contribution of Asp-38 is detected which is, in part, cooperative with that of Tyr-14. Reference spectra of hydrogen-bonded and protonated forms of 19-nortestosterone are reassigned, on the basis of the species identification of D. C. Hawkinson and R. M. Pollack [(1993) Biochemistry 32, 694-698], On the basis of this reassignment, the strength of the 19-nortestosterone polarization produced by the native enzyme is intermediate between complete protonation and the hydrogen-bonding environment of 10 M hydrochloric acid. Since the UVRR spectrum of Tyr-14 is unperturbed upon binding of the steroid, the hydrogen bond to the carbonyl group of 19-nortestosterone may be compensated by a second hydrogen bond to Tyr-14 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 steroid carbonyl group.

Original languageEnglish (US)
Pages (from-to)4441-4447
Number of pages7
JournalBiochemistry
Volume34
Issue number13
DOIs
StatePublished - Sep 1 1995

ASJC Scopus subject areas

  • Biochemistry

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