Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1992 Feb;1(2):259–270. doi: 10.1002/pro.5560010208

Substrate polarization by residues in delta 5-3-ketosteroid isomerase probed by site-directed mutagenesis and UV resonance Raman spectroscopy.

J C Austin 1, A Kuliopulos 1, A S Mildvan 1, T G Spiro 1
PMCID: PMC2142197  PMID: 1339027

Abstract

delta 5-3-Ketosteroid isomerase (KSI: EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by the stereospecific transfer of the steroid 4 beta-proton to the 6 beta-position, using Tyr-14 as a general acid and Asp-38 as a base. Ultraviolet resonance Raman (UVRR) spectra have been obtained for the catalytically active double mutant Y55F + Y88F, which retains Tyr-14 as the only tyrosine residue (referred to as the Y14(0) mutant), and the Y14F mutant, which has 50,000-fold lower activity. The UVRR results establish that binding of the product analog and competitive inhibitors 19-nortestosterone or 4-fluoro-19-nortestosterone to the Y14(0) mutant does not result in the formation of deprotonated Tyr-14. The UVRR spectra of the steroid inhibitors show large decreases in the vinyl and carbonyl stretching frequencies on binding to the Y14(0) enzyme but not on binding to the Y14F enzyme. These changes cannot be mimicked by protonation of the steroids. For 19-nortestosterone, the vinyl and carbonyl stretching frequencies shift down (with respect to the values in aqueous solution) by 18 and 27 cm-1, respectively, on binding to Y14(0) KSI. It is proposed that the changes in the steroid resonance Raman spectrum arise from polarization of the enone moiety via the close proximity of the charged Asp-38 side chain to the vinyl group and the directional hydrogen bond between Tyr-14 and the 3-carbonyl oxygen of the steroid enone. The 230-nm-excited UVRR spectra do not, however, show changes that are characteristic of strong hydrogen bonding from the tyrosine hydrogen. It is proposed that this hydrogen bonding is compensated by a second hydrogen bond to the Tyr-14 oxygen from another protein residue. UVRR spectra of the Y14(0) enzyme obtained using 200 nm excitation show enhancement of the amide II and S Raman bands. The secondary structure of KSI was estimated from the amide II and S intensities and was found to be low in alpha-helical structure. The alpha-helix content was estimated to be in the range of 0-25% (i.e., 10 +/- 15%).

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Copeland R. A., Spiro T. G. Secondary structure determination in proteins from deep (192-223-nm) ultraviolet Raman spectroscopy. Biochemistry. 1987 Apr 21;26(8):2134–2139. doi: 10.1021/bi00382a011. [DOI] [PubMed] [Google Scholar]
  2. Grygon C. A., Spiro T. G. Ultraviolet resonance Raman spectroscopy of distamycin complexes with poly(dA)-poly(dT) and poly(dA-dT): role of H-bonding. Biochemistry. 1989 May 16;28(10):4397–4402. doi: 10.1021/bi00436a041. [DOI] [PubMed] [Google Scholar]
  3. Hawkinson D. C., Eames T. C., Pollack R. M. Kinetic competence of an externally generated dienol intermediate with steroid isomerase. Biochemistry. 1991 Jul 16;30(28):6956–6964. doi: 10.1021/bi00242a021. [DOI] [PubMed] [Google Scholar]
  4. Hildebrandt P. G., Copeland R. A., Spiro T. G., Otlewski J., Laskowski M., Jr, Prendergast F. G. Tyrosine hydrogen-bonding and environmental effects in proteins probed by ultraviolet resonance Raman spectroscopy. Biochemistry. 1988 Jul 26;27(15):5426–5433. doi: 10.1021/bi00415a007. [DOI] [PubMed] [Google Scholar]
  5. Kuliopulos A., Mildvan A. S., Shortle D., Talalay P. Kinetic and ultraviolet spectroscopic studies of active-site mutants of delta 5-3-ketosteroid isomerase. Biochemistry. 1989 Jan 10;28(1):149–159. doi: 10.1021/bi00427a022. [DOI] [PubMed] [Google Scholar]
  6. Kuliopulos A., Mullen G. P., Xue L., Mildvan A. S. Stereochemistry of the concerted enolization catalyzed by delta 5-3-ketosteroid isomerase. Biochemistry. 1991 Apr 2;30(13):3169–3178. doi: 10.1021/bi00227a003. [DOI] [PubMed] [Google Scholar]
  7. Kuliopulos A., Westbrook E. M., Talalay P., Mildvan A. S. Positioning of a spin-labeled substrate analogue into the structure of delta 5-3-ketosteroid isomerase by combined kinetic, magnetic resonance, and X-ray diffraction methods. Biochemistry. 1987 Jun 30;26(13):3927–3937. doi: 10.1021/bi00387a028. [DOI] [PubMed] [Google Scholar]
  8. Weintraub H., Baulieu E. E. Etude de la delta 5-4 3-oxostéroide isomérase. Caractéristiques des groupes impliqués dans le transfert de protons. Eur J Biochem. 1970 Feb;12(2):217–221. doi: 10.1111/j.1432-1033.1970.tb00840.x. [DOI] [PubMed] [Google Scholar]
  9. Xue L. A., Kuliopulos A., Mildvan A. S., Talalay P. Catalytic mechanism of an active-site mutant (D38N) of delta 5-3-ketosteroid isomerase. Direct spectroscopic evidence for dienol intermediates. Biochemistry. 1991 May 21;30(20):4991–4997. doi: 10.1021/bi00234a022. [DOI] [PubMed] [Google Scholar]
  10. Xue L. A., Talalay P., Mildvan A. S. Studies of the mechanism of the delta 5-3-ketosteroid isomerase reaction by substrate, solvent, and combined kinetic deuterium isotope effects on wild-type and mutant enzymes. Biochemistry. 1990 Aug 14;29(32):7491–7500. doi: 10.1021/bi00484a019. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES