The specific, submicromolar-Km ADP-ribose pyrophosphatase purified from human placenta is enzymically indistinguishable from recombinant NUDT9 protein, including a selectivity for Mn2+ as activating cation and increase in Km for ADP-ribose, both elicited by H2O2.
Free ADP-ribose is a putative second messenger and also a potentially toxic compound due to its non-enzymic reactivity towards protein side chains. ADP-ribose hydrolysis is catalysed by NDP-sugar/alcohol pyrophosphatases of differing specificity, including a highly specific, low-K(m) ADP-ribose pyrophosphatase. In humans, a submicromolar-K(m) ADP-ribose pyrophosphatase has been purified from placenta, while recombinant NUDT9 has been described as a similarly specific enzyme with a nudix motif, but with a 10(2)-10(3) higher K(m). Here, a comparative study of both proteins is presented showing that they are in fact enzymically indistinguishable; crucially, they both have submicromolar K(m) for ADP-ribose. This study firmly supports the view that the ADP-ribose pyrophosphatase present in human tissues is a product of the NUDT9 gene. In addition, this study reveals previously unknown properties of both enzyme forms. They display the same, differential properties in the presence of Mg(2+) or Mn(2+) as activating cations with respect to substrate specificity, ADP-ribose saturation kinetics, and inhibition by fluoride. Treatment with H(2)O(2) alters the Mg(2+)/Mn(2+) responses and increases the K(m) values for ADP-ribose, changes that are reversed by DTT. The results are discussed in relation to the proposed roles for ADP-ribose in oxidative/nitrosative stress and for ADP-ribose pyrophosphatase as a protective enzyme whose function is to limit the intracellular accumulation of ADP-ribose.
Carloto
António
A
Unidad de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Extremadura, 06080 Badajoz, Spain.
Costas
María Jesús
MJ
Cameselle
José Carlos
JC
McLennan
Alexander G
AG
Ribeiro
João Meireles
JM
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology*; D010920:Placenta / Q000201:enzymology*; D011755:Pyrophosphatases / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D000246:Adenosine Diphosphate Ribose; D008345:Manganese; D006861:Hydrogen Peroxide; C058796:ADP-ribose pyrophosphatase I; C429463:NUDT9; D011755:Pyrophosphatases; D008274:Magnesium; D004229:Dithiothreitol