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.
D000227:Adenine Nucleotides / Q000378:metabolism*; D000345:Affinity Labels / Q000138:chemical synthesis*; D001665:Binding Sites; D003349:Cortisone Reductase / Q000037:antagonists & inhibitors; D003839:Deoxyadenosines / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D007202:Indicators and Reagents; D007700:Kinetics; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*
D000227:Adenine Nucleotides; D000345:Affinity Labels; D003839:Deoxyadenosines; D007202:Indicators and Reagents; D011506:Proteins; C039744:5'-bromoacetamido-5'-deoxyadenosine; D003349:Cortisone Reductase