An NADH-induced conformational change that mediates the sequential 3β-hydroxysteroid dehydrogenase/isomerase activities is supported by affinity labeling and the time-dependent activation of isomerase

J. L. Thomas, C. Frieden, W. E. Nash, R. C. Strickler

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Abstract

3β-Hydroxysteroid dehydrogenase (3β-HSD) and steroid Δ-isomerase were copurified as a single protein from human placental microsomes. Because NADH is an essential activator of isomerase (Kact = 2.4 μM, Vmax = 0.6 μmol/min/mg), the affinity alkylating nucleotide, 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5′-diphosphate (8-BDB-TADP), was synthesized. 8-BDB-TADP activates isomerase (Kact = 338 μM, Vmax = 2.1 μmol/min/mg) prior to inactivating the enzyme. The inactivation kinetics for isomerase fit the Kitz and Wilson model for time-dependent, irreversible inhibition by 8-BDB-TADP (KI = 314 μM, first order maximal rate constant kobs = 7.8 × 10-3 s-1). NADH (50 μM) significantly protects isomerase from inactivation by 8-BDB-TADP (100 μM). The isomerase activity is inactivated more rapidly by 8-BDB-TADP as the concentration of the affinity alkylator increases from 67 μM (t1/2 = 8.4 min) to 500 μM (t 1/2 = 2.4 min). In sharp contrast, the 3β-HSD activity is inactivated more slowly as the concentration of 8-BDB-TADP increases from 67 μM (t1/2 = 4.8 min) to 500 μM (t1/2 = 60.0 min). We hypothesized that the paradoxical kinetics of 3β-HSD inactivation is a consequence of the activation of isomerase by 8-BDB-TADP via a nucleotide-induced shift in enzyme conformation. Biophysical support for an NADH-induced conformational change was obtained using stopped-flow fluorescence spectroscopy. The binding of NADH (10 μM) quenches the intrinsic fluorescence of the enzyme protein in a time-dependent manner (rate constant kapp = 8.1 × 10-3s-1, t1/2 = 85 s). A time lag is also observed for the activation of isomerase by NADH. This combination of affinity labeling and biophysical data using nucleotide derivatives supports our model for the sequential reaction mechanism; the cofactor product of the 3β-HSD reaction, NADH, activates isomerase by inducing a conformational change in the single, bifunctional enzyme protein.

Original languageEnglish
Pages (from-to)21003-21008
Number of pages6
JournalJournal of Biological Chemistry
Volume270
Issue number36
StatePublished - Sep 8 1995

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