The mechanism of the polynucleotide phosphorylase-catalyzed arsenolysis of ADP.
Using ADP and arsenate (AsV), polynucleotide phosphorylase (PNPase) catalyzes the apparent arsenolysis of ADP to AMP-arsenate and inorganic phosphate, with the former hydrolyzing rapidly into AMP and AsV. However, in the presence of glutathione, AMP-arsenate may also undergo reductive decomposition, yielding AMP and arsenite (AsIII). In order to clarify the mechanism of ADP arsenolysis mediated by Escherichia coli PNPase, we analyzed the time course of the reaction in the presence of increasing concentrations of ADP, with or without polyadenylate (poly-A) supplementation. These studies revealed that increasing supply of ADP enhanced the consumption of ADP but inhibited the production of both AMP and AsIII. Formation of these products was amplified by adding trace amount of poly-A. Furthermore, AMP and AsIII production accelerated with time, whereas ADP consumption slowed down. These observations collectively suggest that PNPase does not catalyze the arsenolysis of ADP directly (in a single step), but in two separate consecutive steps: the enzyme first converts ADP into poly-A, then it cleaves the newly synthesized poly-A by arsenolysis. It is inferred that one active site of PNPase can catalyze only one of these reactions at a time and that high ADP concentrations favor poly-A synthesis, thereby inhibiting the arsenolysis.
Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, Szigeti út 12, Pécs, Hungary.
Balázs
B
Németi
Maria Elena
ME
Regonesi
Paolo
P
Tortora
Zoltán
Z
Gregus
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D018053:Arsenites / Q000378:metabolism; D055162:Biocatalysis; D004926:Escherichia coli / Q000201:enzymology; D011061:Poly A / Q000378:metabolism; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism*
D018053:Arsenites; D011061:Poly A; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D011117:Polyribonucleotide Nucleotidyltransferase; C015001:arsenite