Analysis of coenzyme binding by human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase using 5'-[p-(fluorosulfonyl)benzoyl]adenosine, an affinity labeling cofactor analog.
3 beta-Hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase copurify as a single, homogeneous protein from human placental microsomes. Affinity alkylation with 2 alpha-bromoacetoxyprogesterone suggests that the dehydrogenase and isomerase substrate steroids bind at different sites on the same protein. However, the coenzyme, NADH, completely abolishes the alkylation of both enzyme activities by the progestin analog [Thomas J .L., Myers R. P., Rosik L. O. and Strickler R. C., J. Steroid Biochem. 36 (1990) 117-123]. Unlike bacterial 3-keto-5-ene-steroid isomerase, the human isomerase reaction is stimulated by diphosphopyridine nucleotides (NADH, NAD+). The affinity labeling nucleotide analog, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSA), inactivates the dehydrogenase and isomerase activities at similar rates in an irreversible manner which follows first order kinetics with respect to both time and alkylator concentration (0.2-0.6 mM). FSA is a cofactor site-directed reagent that binds with similar affinity as a competitive inhibitor of NAD+ reduction by dehydrogenase (Ki = 162 microM) or as a stimulator of isomerase (Km = 153 microM). Parallel plots derived from Kitz and Wilson analysis indicate that FSA inactivates the two enzyme activities with equal alkylation efficiency (k3/Ki = 1/slope = 0.51/mol-s for both). The 3 beta-hydroxysteroid substrate, pregnenolone, protects isomerase as well as dehydrogenase from inactivation by FSA. These observations are evidence for a single cofactor binding region which services both enzyme activities.
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progestin 3-keto-5-ene-steroid diphosphopyridine nucleotides NADH NAD+ nucleotide 5'-[p-(fluorosulfonyl)benzoyl]adenosine FSA 5'-[p-(fluorosulfonyl)benzoyl]adenosine 3 beta-hydroxy-5-ene-steroid 3 beta-Hydroxy-5-ene-steroid 3 beta-Hydroxy-5-ene-steroid dehydrogenase dehydrogenase dehydrogenase dehydrogenase steroid 5----4-ene-isomerase 3-keto-5-ene-steroid isomerase dehydrogenase human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase steroid 5----4-ene-isomerase NAD+ FSA 3 beta-hydroxysteroid pregnenolone FSA alpha-bromoacetoxyprogesterone steroids NADH