Metal ions binding to NAD-glycohydrolase from the venom of Agkistrodon acutus: Regulation of multicatalytic activity

Abstract

AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu²⁺ ions that are essential for its multicatalytic activity. In this study, the interactions between divalent metal ions and AA-NADase and the effects of metal ions on its structure and activity have been investigated by equilibrium dialysis, isothermal titration calorimetry, fluorescence, circular dichroism, dynamic light scattering and HPLC. The results show that AA-NADase has two classes of Cu²⁺ binding sites, one activator site with high affinity and approximately six inhibitor sites with low affinity. Cu²⁺ ions function as a switch for its NADase activity. In addition, AA-NADase has one Mn²⁺ binding site, one Zn²⁺ binding site, one strong and two weak Co²⁺ binding sites, and two strong and six weak Ni²⁺ binding sites. Metal ion binding affinities follow the trend Cu²⁺ > Ni²⁺ > Mn²⁺ > Co²⁺ > Zn²⁺, which accounts for the existence of one Cu²⁺ in the purified AA-NADase. Both NADase and ADPase activities of AA-NADase do not have an absolute requirement for Cu²⁺, and all tested metal ions activate its NADase and ADPase activities and the activation capacity follows the trend Zn²⁺ > Mn²⁺ > Cu²⁺ ∼ Co²⁺ > Ni²⁺. Metal ions serve as regulators for its multicatalytic activity. Although all tested metal ions have no obvious effects on the global structure of AA-NADase, Cu²⁺- and Zn²⁺-induced conformational changes around some Trp residues have been observed. Interestingly, each tested metal ion has a very similar activation of both NADase and ADPase activities, suggesting that the two different activities probably occur at the same site.