Small-molecule reductants inhibit multicatalytic activity of AA-NADase from Agkistrodon acutus venom by reducing the disulfide-bonds and Cu(II) of enzyme.
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(II) and has disulfide-bond linkages between two peptide chains. The effects of the reduction of the disulfide-bonds and Cu(II) in AA-NADase by small-molecule reductants on its NADase and ADPase activities have been investigated by polyacrylamide gel electrophoresis, high performance liquid chromatography, electron paramagnetic resonance spectroscopy and isothermal titration calorimetry. The results show that AA-NADase has six disulfide-bonds and fifteen free cysteine residues. L-ascorbate inhibits AA-NADase on both NADase and ADPase activities through the reduction of Cu(II) in AA-NADase to Cu(I), while other reductants, dithiothreitol, glutathione and tris(2-carboxyethyl)phosphine inhibit both NADase and ADPase activities through the reduction of Cu(II) to Cu(I) and the cleavage of disulfide-bonds in AA-NADase. Apo-AA-NADase can recover its NADase and ADPase activities in the presence of 1 mM Zn(II). However, apo-AA-NADase does not recover any NADase or ADPase activity in the presence of 1 mM Zn(II) and 2 mM TCEP. The multicatalytic activity relies on both disulfide-bonds and Cu(II), while Cu(I) can not activate the enzyme activities. AA-NADase is probably only active as a dimer. The inhibition curves for both ADPase and NADase activities by each reductant share a similar trend, suggesting both ADPase and NADase activities probably occur at the same site. In addition, we also find that glutathione and L-ascorbate are endogenous inhibitors to the multicatalytic activity of AA-NADase.
Department of Chemistry, University of Science and Technology of China, Hefei 230026, People's Republic of China.
Li-Yun
LY
Zhang
Xiao-Long
XL
Xu
Zhao-Feng
ZF
Luo
Hao
H
Wu
Deng-Ke
DK
Shen
Li-Li
LL
Peng
Yang-Zhong
YZ
Liu
D000818:Animals; D003300:Copper / Q000737:chemistry; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors* / Q000201:enzymology*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D009244:NAD+ Nucleosidase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D010084:Oxidation-Reduction; D010720:Phosphines / Q000494:pharmacology
D003300:Copper; D003435:Crotalid Venoms; D003545:Cysteine; D004220:Disulfides; D004229:Dithiothreitol; D005978:Glutathione; D009244:NAD+ Nucleosidase; D010720:Phosphines; C031442:Agkistrodon venoms; C080938:tris(2-carboxyethyl)phosphine