Abstract
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.
Highlights
This compares with a K i value of 10 mM for adenosine under similar excated kineticsof the enzyme inactivation which are associated with affinity labeling [6, 7, 9]
The enzyme inactivation reaction proceeded reacts with an amino acid at an adenine nucleotide binding by a first order kinetic process
The results suggest that 5‘-BADA inactivates proteins because they modify amino acids to form known the enzyme by irreversibly binding to the adenindeo- carboxymethyl derivatives
Summary
A Dixon plotof data from enzymekinetic measurements showed that 5‘-BADA is a competitiveinhibitor of NADH oxidation by 3a,20&hydroxysteroid dehydrogenase witha K i value of 11.8mM This compares with a K i value of 10 mM for adenosine under similar excated kineticsof the enzyme inactivation which are associated with affinity labeling [6, 7, 9]. The present report describes the synthesis and characterization of 5’-BADA and its testing witha NADH-dependent enzyme This novel nucleoside is the first member of a class of affinity alkylating adenosine analogs to contain a bromoacetamido side chain on the furanoside moiety of adenosine. Selectively labeling amino acid residueast theadenine 5’-BADA wasdesigned to be highly soluble andstable in nucleotide binding sites of enzymatic and regulatory water and to forma carboxymethylamino acidderivative proteins. Organisms contain great numbers of biochemical redox reactions which are promoted by oxidoreductase enzymes inter-
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