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The delta 5-3-ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni 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 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 delta 5-3-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 Y14F 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].(ABSTRACT TRUNCATED AT 250 WORDS)

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Ultraviolet resonance Raman (UVRR) spectra, with 260-nm excitation, are reported for oxidized and reduced nicotinamide adenine dinucleotides (NAD+ and NADH, respectively). Corresponding spectra are reported for these coenzymes when bound to the enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and liver and yeast alcohol dehydrogenases (LADH and YADH). The observed differences between the coenzyme spectra are interpreted in terms of conformation, hydrogen bonding, and general environment polarity differences between bound and free coenzymes and between coenzymes bound to different enzymes. The possibility of adenine protonation is discussed. UVRR spectra with 220-nm excitation also are reported for holo- and apo-GAPDH (GAPDH-NAD+ and GAPDH alone, respectively). In contrast with the 260-nm spectra, these show only bands due to vibrations of aromatic amino acid residues of the protein. The binding of coenzyme to GAPDH has no significant effect on the aromatic amino acid bands observed. This result is discussed in the light of the known structural change of GAPDH on binding coenzyme. Finally, UVRR spectra with 240-nm excitation are reported for GAPDH and an enzyme-substrate intermediate of GAPDH. Perturbations are reported for tyrosine and tryptophan bands on forming the acyl enzyme.