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

James L Thomas,Carl Frieden,William E Nash,Ronald C Strickler

doi:10.1074/jbc.270.36.21003

James L Thomas, Carl Frieden + Show 2 more

Open Access

https://doi.org/10.1074/jbc.270.36.21003

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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.

Highlights

§ To whom correspondence should be addressed: Dept. of Obstetrics and Gynecology, Washington University School of Medicine, 4911 Barnes Hospital Plaza, St
Evidence That 8-BDB-TADP Is a Site-directed Alkylator of Isomerase—Like NADH, 8-BDB-TADP activated isomerase according to Michaelis-Menton kinetics (Table I)
The Kact and Vmax values reported for 8-BDB-TADP were calculated from initial velocities (Ͻ1.0 min) because isomerase was measurably inactivated within 1–3 min by the higher concentrations of the affinity alkylator

Summary

EXPERIMENTAL PROCEDURES

Materials—Steroid hormones, pyridine nucleotides, ADP, and DEAE-Sephadex were purchased from Sigma; 5-pregnene-3,20-dione and 5-androstene-3,17-dione were from Steraloids Inc. (Wilton, NH); reagent grade salts and analytical grade solvents were from Fisher; 1,4-dibromo-2,3-butanedione was from Aldrich; AG50W-X4(Hϩ) ion exchange gel was from Bio-Rad. The control and experimental mixtures contained the same concentration of the potentially protecting steroid or cofactor with no increase in final solvent content compared to incubations without protector The concentrations of these ligands were at least three times the Km or KI measured for 3␤-HSD or isomerase activity to facilitate competition with a subsaturating concentration of 8-BDB-TADP (100.0 ␮M). 1) Isomerase substrate (5-pregnene-3,20-dione, at Km ϭ 10.0 ␮M) was preincubated with the enzyme for 2.0 min, and NADH (at Kact ϭ 2.4 ␮M) was added to start the reaction at zero time. In the spectrophotometric assay (241 nm) for isomerase activity (5), the substrate or NADH was quickly mixed (Ͻ5 s) in the cuvette at zero time (after the 2.0-min preincubation) so that the nmol progesterone formed could be measured from the Varian Cary 219 recorder tracing. The actual concentration of enzyme protein was determined by ultraviolet spectral measurements in 6.0 N guanidine (34 Tyr plus 16 Trp residues/enzyme dimer yielded a molar extinction coefficient at 280 nm of 1.346 ϫ 105/dimer)

RESULTS

Nucleotide analog

DISCUSSION

REACTION II