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  • Title: An NADH-induced conformational change that mediates the sequential 3 beta-hydroxysteroid dehydrogenase/isomerase activities is supported by affinity labeling and the time-dependent activation of isomerase.
    Author: Thomas JL, Frieden C, Nash WE, Strickler RC.
    Journal: J Biol Chem; 1995 Sep 08; 270(36):21003-8. PubMed ID: 7673125.
    Abstract:
    3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and steroid delta-isomerase were copurified as a single protein from human placental microsomes. Because NADH is an essential activator of isomerase (Kact = 2.4 microM, Vmax = 0.6 mumol/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 microM, Vmax = 2.1 mumol/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 microM, first order maximal rate constant kobs = 7.8 x 10(-3) s-1). NADH (50 microM) significantly protects isomerase from inactivation by 8-BDB-TADP (100 microM). The isomerase activity is inactivated more rapidly by 8-BDB-TADP as the concentration of the affinity alkylator increases from 67 microM (t1/2 = 8.4 min) to 500 microM (t1/2 = 2.4 min). In sharp contrast, the 3 beta-HSD activity is inactivated more slowly as the concentration of 8-BDB-TADP increases from 67 microM (t1/2 = 4.8 min) to 500 microM (t1/2 = 60.0 min). We hypothesized that the paradoxical kinetics of 3 beta-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 microM) quenches the intrinsic fluorescence of the enzyme protein in a time-dependent manner (rate constant kapp = 8.1 x 10(-3) s-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 3 beta-HSD reaction, NADH, activates isomerase by inducing a conformational change in the single, bifunctional enzyme protein.
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