Adenosine 5'-(2-bromoethyl)-phosphate. A new affinity label for adenine nucleotide sites in proteins

A new adenosine analogue adenosine 5'-(2-bromoethyl)-phosphate has been synthesized. The reactive moiety, a bromoalkyl group, has the ability to react with the nucleophilic side chains of several amino acids. This compound reacts with NAD-dependent isocitrate dehydrogenase from pig heart, causing inactivation. Addition of the allosteric regulator ADP to the reaction mixture protects the enzyme from loss of activity. A second adenosine analogue has been synthesized, adenosine 5'-(n-propyl)-phosphate, which is used to assess any effects that might arise from the noncovalent interaction of adenosine 5'-(2-bromoethyl)-phosphate with the enzyme. It is proposed that adenosine 5'-(2-bromoethyl)-phosphate reacts with an adenine nucleotide site on isocitrate dehydrogenase and that this compound may have general applicability as an affinity label of catalytic and regulatory adenine nucleotide sites in proteins.