title: Cu(ii)- and disulfide bonds-induced stabilization during the guanidine hydrochloride- and thermal-induced denaturation of NAD-glycohydrolase from the venom of Agkistrodon acutus.
abstract: NAD-glycohydrolase (AA-NADase) from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase-like activities. Among all identified NADases, only AA-NADase is a disulfide-linked dimer and contains Cu(2+). Cu(2+) and disulfide bonds are essential for its multicatalytic activity. In this study, the effects of Cu(2+) and disulfide-bonds on guanidine hydrochloride (GdnHCl)- and thermal-induced unfolding of AA-NADase have been investigated by fluorescence, circular dichroism (CD) and differential scanning calorimetry (DSC). Cu(2+) and disulfide bonds not only increase the free energy change during the GdnHCl-induced unfolding as determined by fluorescence, but also increase the overall enthalpy change and the transition temperature during the thermal-induced unfolding as determined by CD and DSC. The slope of the GdnHCl-induced unfolding curve at its midpoint and the heat capacity of thermal-induced unfolding are slightly affected by Cu(2+) but significantly decrease after reduction of three disulfide-bonds. This work suggests that Cu(2+) stabilizes the folded state by increasing the enthalpy of unfolding, while disulfide-bonds stabilize the folded state by increasing the enthalpy of unfolding and stabilizing the packing of hydrophobic residues. Thus both Cu(2+) and disulfide bonds play a structural role in its multicatalytic activity.
authors: Department of Chemistry, University of Science and Technology of China, Hefei, 230026, PR China.
authors: Liyun
authors: L
authors: Zhang
authors: Xiaolong
authors: X
authors: Xu
authors: Zhaofeng
authors: Z
authors: Luo
authors: Yan
authors: Y
authors: Zhang
authors: Dengke
authors: D
authors: Shen
authors: Lili
authors: L
authors: Peng
authors: Jiajia
authors: J
authors: Song
mesh_terms: D017836:Agkistrodon; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002413:Cations, Divalent / Q000737:chemistry; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry*; D003435:Crotalid Venoms / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D019791:Guanidine / Q000494:pharmacology; D009244:NAD+ Nucleosidase / Q000737:chemistry*; D010720:Phosphines / Q000494:pharmacology; D011489:Protein Denaturation; D055550:Protein Stability; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D002413:Cations, Divalent; D003300:Copper; D003435:Crotalid Venoms; D004220:Disulfides; D019791:Guanidine; D009244:NAD+ Nucleosidase; D010720:Phosphines; C080938:tris(2-carboxyethyl)phosphine