Positioning of a spin-labeled substrate analog into the structure of .DELTA.5-3-ketosteroid isomerase by combined kinetic, magnetic resonance, and x-ray diffraction methods

Abstract

We have shown by kinetic and magnetic resonance measurements that a spin-labeled substrate analogue, spiro[doxyl-2,3'-5' alpha-androstan]-17'beta-ol, binds at the substrate site of crystalline delta 5-3-ketosteroid isomerase (steroid delta-isomerase; EC 5.3.3.1) of Pseudomonas testosteroni. The spin-labeled steroid is a linear competitive inhibitor with a Ki value (25 +/- 5 microM) that is consistent with dissociation constants obtained by direct binding measurements based on changes in the electron paramagnetic resonance spectrum of the nitroxide, longitudinal relaxation rates of water protons, and longitudinal and transverse relaxation rates of carbon-bound protons of the isomerase. These binding studies yield a stoichiometry for the nitroxide of 1 per subunit of the enzyme. Measurements of the longitudinal relaxation rates of water protons indicate that the 3-doxyl portion of the spin-label is highly immobilized yet is exposed to solvent. Paramagnetic effects of the nitroxide on T1 defined distances to several previously assigned [Benisek, W. F., & Ogez, J. R. (1982) Biochemistry 21, 5816-5825] and newly assigned protons of the enzyme. These distances were then used to locate (with an accuracy of +/- 2 A) the nitroxide moiety at a unique position in a partially refined 2.5-A resolution X-ray structure of native isomerase. Three of five additional proton resonance peaks, attributed to ring-shielded methyl groups, could be assigned to specific residues on the basis of distances from the spin-label in the X-ray structure. The remaining portion of the spin-labeled steroid was then docked into the X-ray structure in a hydrophobic cavity of the enzyme. This position of the steroid is consistent with the steroid binding site previously proposed [Westbrook, E. M., Piro, O. E., & Sigler, P. B. (1984) J. Biol. Chem. 259, 9096-9103]. However, the rotational orientation of this steroid about its long axis could not be unambiguously established. If we assume that steroid substrates and the spin-labeled inhibitor bind to the same site, but with reversal of the 3- and 17-positions, then the phenolic hydroxyl of Tyr-55 is optimally positioned to function as the general acid that protonates the 3-keto group of the substrate, facilitated by the negative end of the dipole of a 10-residue alpha-helix, the only helix in the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Substrate polarization by residues in Δ5‐3‐ketosteroid isomerase probed by site‐directed mutagenesis and UV resonance Raman spectroscopy

“…10%) ahelical content in KSI. Although this value agrees with the helix content of 8% observed in the X-ray structure of KSI (Kuliopulos et al, 1987b), the relatively low perchlorate intensity (the protein concentration is higher than usually employed, 136 pM) and lack of correction for detector response makes the estimate prone to errors in the range of f 15% (i.e., helix content is between 0 and 25%). Further evidence in support of low a-helical content in KSI can be drawn from the positions of the amide I11 (1237 cm") and amide I (1667 cm") bands, which are at frequencies that are very indicative of a mixture of &sheet and random structures (Tu, 1986).…”

“…If the same procedure is applied to the Y14F-19-nortestosterone data, a negative deviation of vcZc is again seen, although it is smaller (5 cm"), and the effective AN is 60. This value of AN is larger than that of H 2 0 (AN = 55), a result that is quite surprising when one considers that the active site environment is decidedly hydrophobic, with Phe-82, Phe-86, Phe-100, and Val-74 all in close proximity to the steroid (Kuliopulos et al, 1987b), and with Phe replacing Tyr-14 at the carbonyl group. The Asp-38 side chain (which is presumably deprotonated) is, however, close to the vinyl group, placed to act as the base (B, Fig.…”

“…1) or stepwise process, the latter involving either a dienolate or carbonium ion intermediate. Many of the insights into the catalytic action of KSI have resulted from spectroscopic and structural studies of inhibitor-enzyme complexes (Kuliopulos et al, 1987b(Kuliopulos et al, , 1989Wang et al, 1963) and most recently, such studies have been enhanced by the use of site-directed mutants. In particular, the complexes of KSI and KSI mutants with the competitive inhibitor and product analog 19-nortestosterone have been Proposed reaction scheme for ketosteroid isomerase .…”

The Role of Glutathione in the Isomerization of Δ5-Androstene- 3,17-dione Catalyzed by Human Glutathione Transferase A1-1

“…Its catalytic mechanism has been studied in great detail. A key residue of the active site is Asp 38 , the carboxylate that shuttles a proton from C4 to C6 of the substrate AD in the isomerization reaction (45). A tyrosine residue, Tyr 14 , assisted by Asp 99 promotes the isomerization by hydrogen bonding and polarization of the 3-keto group of AD (46,47).…”

Cloning, nucleotide sequence, and overexpression of the gene coding for delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B

“…Three-dimensional structure of the KSI is known at 6-A (0.6-nm; 1 A = 0.1 nm) resolution (21). A preliminary, unrefined model has been obtained on the basis of a 2.5-A resolution electron density map (9). However, this model possesses an unacceptably high R-factor, and efforts to refine this structure in order to lower the R-factor have not been successful (hla).…”