6-[(4-Bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-monophosphate and 5'-diphosphate: new affinity labels for purine nucleotide sites in proteins

Abstract

Two new adenine nucleotide analogues have been synthesized and characterized: 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-monophosphate and 5'-diphosphate. The bromoketo and dioxobutyl moieties have the ability to react with the nucleophilic side chains of several amino acids, as well as with arginine. 6-[(4-Bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-monophosphate reacts irreversibly with rabbit muscle pyruvate kinase, causing inactivation. Addition of ADP to the reaction mixture (in the presence of Mg2+) markedly decreases the rate of inactivation. Pig heart NAD-dependent isocitrate dehydrogenase is allosterically activated by ADP, which reduces the Km for isocitrate. 6-[(4-Bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate reacts irreversibly with isocitrate dehydrogenase, causing, rapidly, a loss of the ability of ADP to increase the initial velocity of assays conducted at low isocitrate concentrations and, more slowly, inactivation. Addition of ADP to the reaction mixture (in the presence of Mn2+) protects this enzyme against the loss of allosteric activation. It is proposed that the 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenine nucleotides react at the active site of pyruvate kinase and at the ADP activating site of isocitrate dehydrogenase and that these compounds may have general applicability as affinity labels of catalytic and regulatory adenine nucleotide sites in proteins.