Metal ions binding to NAD-glycohydrolase from the venom of Agkistrodon acutus: regulation of multicatalytic activity.
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 Cu(2+) 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 Cu(2+) binding sites, one activator site with high affinity and approximately six inhibitor sites with low affinity. Cu(2+) ions function as a switch for its NADase activity. In addition, AA-NADase has one Mn(2+) binding site, one Zn(2+) binding site, one strong and two weak Co(2+) binding sites, and two strong and six weak Ni(2+) binding sites. Metal ion binding affinities follow the trend Cu(2+) > Ni(2+) > Mn(2+) > Co(2+) > Zn(2+), which accounts for the existence of one Cu(2+) in the purified AA-NADase. Both NADase and ADPase activities of AA-NADase do not have an absolute requirement for Cu(2+), and all tested metal ions activate its NADase and ADPase activities and the activation capacity follows the trend Zn(2+) > Mn(2+) > Cu(2+) ~Co(2+) > Ni(2+). 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, Cu(2+)- and Zn(2+)-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.
Department of Chemistry, University of Science and Technology of China, Hefei, P. R. China. xuxl@ustc.edu.cn
Xiaolong
X
Xu
Liyun
L
Zhang
Zhaofeng
Z
Luo
Dengke
D
Shen
Hao
H
Wu
Lili
L
Peng
Jiajia
J
Song
Yan
Y
Zhang
D017836:Agkistrodon; D000818:Animals; D001665:Binding Sites; D055162:Biocatalysis; D001681:Biological Assay; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D003435:Crotalid Venoms / Q000737:chemistry* / Q000201:enzymology*; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D009532:Nickel / Q000737:chemistry / Q000378:metabolism
D003435:Crotalid Venoms; D008670:Metals; D008345:Manganese; D003300:Copper; D009532:Nickel; D009244:NAD+ Nucleosidase