5'-bromoacetamido-5'-deoxyadenosine. A novel reagent for labeling adenine nucleotide sites in proteins.

We have synthesized and characterized 5'-bromoacetamido-5'-deoxyadenosine (5'-BADA), a new reagent for labeling adenine nucleotide binding sites in enzymatic and regulatory proteins. 5'-BADA possessed exceptionally high solubility and stability in aqueous buffers between pH 5.0 and 8.6 at 25 degrees C. A Dixon plot of data from enzyme kinetic measurements showed that 5'-BADA is a competitive inhibitor of NADH oxidation by 3 alpha,20 beta-hydroxysteroid dehydrogenase with a Ki value of 11.8 mM. This compares with a Ki value of 10 mM for adenosine under similar experimental conditions. Incubating 5'-BADA with a 3 alpha,20 beta-hydroxysteroid dehydrogenase at pH 7.0 and 25 degrees C caused simultaneous loss of both 3 alpha and 20 beta activity. The enzyme inactivation reaction proceeded by a first order kinetic process. The rates of enzyme inactivation as a function of 5'-BADA concentration obeyed saturation kinetics. 2-Bromoacetamide, at ten times the maximum concentration of 5'-BADA, had no measurable effect on enzyme activity during 25 h of incubation. NADH and AMP protected 3 alpha,20 beta-hydroxysteroid dehydrogenase against inactivation by 5'-BADA. The results suggest that 5'-BADA inactivates the enzyme by irreversibly binding to the adenine domain of the NADH cofactor binding region at the catalytic site of 3 alpha,20 beta-hydroxysteroid dehydrogenase. Irreversible binding follows from an alkylation reaction between the bromoacetamido side chain of 5'-BADA and an amino acid at or near the enzyme catalytic site. 5'-BADA is presented as a new reagent for selectively labeling amino acid residues at the adenine nucleotide binding sites of enzymatic and regulatory proteins.

Nicotinamide adenine dinucleotide binding and promotion of enzyme activity: model based on affinity labeling of 3 alpha, 20 beta-hydroxysteroid dehydrogenase with a nucleoside.

5'-[p-(Fluorosulfonyl)benzoyl]adenosine (FSA) was used to affinity-label the NADH binding region of 3 alpha, 20 beta-hydroxysteroid dehydrogenase (3 alpha, 20 beta-HSD) to further test our hypothesis [Sweet, F., & Samant, B. R. (1980) Biochemistry 19, 978-986] that 3 alpha and 20 beta activities occur at the same active site. Incubation of 3 alpha, 20 beta-HSD (0.45 microM) with FSA (125 microM) at pH 7.0 and 0 degrees C caused simultaneous loss of 3 alpha and 20 beta activities by a first-order kinetic process, with t1/2 = 300 min for both activities. Dinucleotides and adenosine mononucleotides which acted as competitive inhibitors protected 3 alpha, 20 beta-HSD against inactivation by FSA in a concentration-dependent manner, in the order reduced nicotinamide dinucleotide phosphate greater than oxidized nicotinamide dinucleotide phosphate greater than adenosine diphosphate-ribose greater than adenosine diphosphate greater than adenosine monophosphate (AMP) greater than adenosine. Oxidized and reduced nicotinamide mononucleotides (NMH and NMNH) and steroid substrates did not protect 3 alpha, 20 beta-HSD against affinity labeling by FSA. Although NMN was not a competitive inhibitor of 3 alpha, 20 beta-HSD, NMN with AMP and also AMP with NMNH produced positive cooperativity for competitive inhibition of 3 alpha, 20 beta-HSD. The results from FSA affinity labeling of the cofactor region confirm that both 3 alpha and 20 beta activities share the same active site of 3 alpha, 20 beta-HSD and suggest a model of cofactor binding and promotion of enzyme activity. The adenosine 5'-phosphate component anchors the NAD or NADH to an adenosine domain in the cofactor binding region. The nicotinamide nucleotide component then carries out the hydrogen-transfer reaction at a neighboring domain near the steroid binding region.

Affinity labeling of 3 alpha, 20 beta-hydroxysteroid dehydrogenase with a nucleoside analog.

Incubation of 3 alpha, 20 beta-hydroxysteroid dehydrogenase (3 alpha, 20 beta-HSD; E.C.1.1.1.53) with the nucleoside 5'-p-fluorosulfonylbenzoyladenosine (FSA) caused a time-dependent and irreversible loss in enzyme activity. Both 3 alpha- and 20 beta-hydroxysteroid oxidoreductase activities decreased at equal rates by a first order kinetic process (in 0.05M phosphate buffer at pH 6.0 and 25 degrees C, t1/2 = 170 min). Incubation of 3 alpha, 20 beta-HSD was quenched by addition of 2-mercaptoethanol which instantaneously reacts with the fluorosulfonyl group of FSA. The cofactor NADH protected 3 alpha, 20 beta-HSD against inactivation by FSA, in a concentration-dependent manner. However, progesterone did not protect 3 alpha, 20 beta-HSD against inactivation by FSA. Evidently, FSA causes inactivation of the enzyme by irreversibly binding to the NADH-binding region at the active site of 3 alpha, 20 beta-HSD. Both 3 alpha- and 20 beta-hydroxysteroid oxidoreductase activities disappeared at equal rates under a variety of enzyme-inactivating conditions. These results suggest that both 3 alpha- and 20 beta-activities occur at the same active site of 3 alpha, 20 beta-HSD.

Synthesis of medroxyprogesterone bromoacetate for affinity labeling.

Medroxyprogesterone bromoacetate (17alpha-hydroxy-6alpha-methyl-4-pregnene-3,20-dione 17-bromoacetate) was synthesized by reaction of 17alpha-hydroxy-6alpha-methyl-4-pregnene-3,20-dione with bromoacetic acid--trifluoroacetic anhydride followed by treatment of the intermediate with dilute ethanolic HBr. The product forms conjugates with L-cysteine, L-histidine, and L-methionine and inactivates 20beta-hydroxy steroid dehydrogenase (E.C. 1.1.1.53.) from Streptomyces hydrogenans in a time-dependent and irreversible manner. The title compound possesses a long-acting progestational effect in day 9 pregnant bilaterally ovariectomized rats. The affinity labeling analogue of the oral contraceptive medroxyprogesterone acetate is proposed for use in reproductive biological experiments.