Positioning of a Spin-Labeled Substrate Analogue into the Structure of A5-3-Ketosteroid Isomerase by Combined Kinetic, Magnetic Resonance, and X-ray Diffraction Methods

Athan Kuliopulos, Albert S. Mildvan, Edwin M. Westbrook, Paul Talalay

Research output: Contribution to journalArticlepeer-review

Abstract

We have shown by kinetic and magnetic resonance measurements that a spin-labeled substrate analogue, spiro[doxyl-2, 3/-5/α-androstan]-17/ß-ol, binds at the substrate site of crystalline A5-3-ketosteroid isomerase (steroid A-isomerase; EC 5.3.3.1) of Pseudomonas testosteroni. The spin-labeled steroid is a linear competitive inhibitor with a value (25 ± 5 µM) that is consistent with dissociation constants obtained by direct binding measurements based on changes in (a) the electron paramagnetic resonance spectrum of the nitroxide, (b) longitudinal relaxation rates of water protons, and (c) 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 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 a-helix, the only helix in the molecule. In this proposed binding mode, the 4- and 6-positions of the steroid substrate are near Asp-38 and Asn-57, suggesting that Asp-38 functions as the general base. Thus, the present results not only explain the reaction mechanism of steroid isomerase but are also consistent with the sites of covalent modification of the enzyme by affinity labels and mechanism-based inhibitors.

Original languageEnglish (US)
Pages (from-to)3927-3937
Number of pages11
JournalBiochemistry
Volume26
Issue number13
DOIs
StatePublished - Jan 1 1987

ASJC Scopus subject areas

  • Biochemistry

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