Periodate-oxidized 3-aminopyridine adenine dinucleotide phosphate as a fluorescent affinity label for pigeon liver malic enzyme

Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate resulted in oxidation of the ribose linked to 3-aminopyridine ring and cleavage of the dinucleotide into 3-aminopyridine and adenosine moieties. These two moieties were separated by thin layer chromatography and were synergistically bound to pigeon liver malic enzyme (EC 1.1.1.40), causing inactivation of the enzyme. The inactivation showed saturation kinetics. The apparent binding constant for the reversible enzyme-reagent binary complex (K(I)) and the maximum inactivation rate constant at saturating reagent concentration (k(max)) were found to be 1.1 ± 0.02 mM and 0.068 ± 0.001 min^-1, respectively. L-Malate at low concentration enhanced the inactivation rate by lowering the K(I) value whereas high malate concentration increased the k(max). Mn²⁺ or NADP⁺ partially protected the enzyme from the inactivation and gave additive protection when used together. L-Malate eliminated the protective effect of NADP⁺ or Mn²⁺. Maximum and synergistic protection was afforded by NADP⁺, Mn²⁺ plus L-malate (or tartronate). Oxidized and cleaved 3-aminopyridine adenine dinucleotide phosphate was also found to be a competitive inhibitor versus NADP⁺ in the oxidative decarboxylation reaction catalyzed by malic enzyme with a K(i) value of 4.1 ± 0.1 μM. 3-Aminopyridine adenine dinucleotide phosphate or its periodate-oxidized cleaved products bound to the enzyme anticooperatively. Oxidized 3-aminopyridine adenine dinucleotide phosphate labeled the nucleotide binding site of the enzyme with a fluorescent probe which may be readily traced or quantified. The completely inactivated enzyme incorporated 2 mol of reagent/mol of enzyme tetramer. The inactivation was partially reversible by dilution and could be made irreversible by treating the modified enzyme with sodium borohydride. This fluorescent compound and its counterpart-oxidized 3-aminopyridine adenine dinucleotide may be a potential affinity label for all other NAD(P)⁺-dependent dehydrogenases.