On the Number of Steroid‐Binding Sites of Δ5‐3‐Oxosteroid Isomerase

Trevor M. PENNING, Edwin M. WESTBROOK, Paul TALALAY

Research output: Contribution to journalArticle

Abstract

The number of steroid‐binding sites in Δ5‐3‐oxosteroid isomerase of Pseudomonas testosteroni (EC 5.3.3.1) has been determined from measurements of the red shift of the ultraviolet chromo‐phore of 19‐nortestosterone upon binding to the enzyme. The experiments include spectroscopic measurements when limiting concentrations of either 19‐nortestosterone or isomerase are titrated with varying concentrations of the complementary ligand. Analysis of the results indicates one binding site per subunit (Mr 13394). Scatchard plots indicate a single family of equivalent binding sites. 5,10‐Secoestr‐5‐yne‐3,10,17‐trione is a suicide substrate of isomerase [Batzold & Robinson (1975) J. Am. Chem. Soc. 97, 2576]. The time course for inactivation of isomerase with an excess of 5,10‐seco[7‐3H]estr‐5‐yne‐3,10,17‐trione was parallel to the covalent incorporation of steroid and gave a final stoichiometry of nearly one steroid molecule per subunit of enzyme. Alkylation of [14C]iso‐merase with excess of this 3H‐labeled steroid followed by gel‐filtration and dialysis gave an inactivated enzyme with a 3H/14C ratio that corresponds to one molecule of steroid bound per subunit; this stoichiometry was constant over a wide range of protein concentrations (0.1 − 10 mg/ml). Diffusion of [3H]progesterone into hexagonal crystals of isomerase showed that at saturation one steroid molecule was bound per protomer. Taken together these findings strongly support the conclusion that one molecule of steroid is bound per subunit of isomerase both in solution and in the crystal state.

Original languageEnglish (US)
Pages (from-to)461-469
Number of pages9
JournalEuropean Journal of Biochemistry
Volume105
Issue number3
DOIs
StatePublished - Apr 1980

ASJC Scopus subject areas

  • Biochemistry

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