Mechanism of the Escherichia coli ADP-ribose pyrophosphatase, a Nudix hydrolase.
Escherichia coli ADP-ribose (ADPR) pyrophosphatase (ADPRase), a Nudix enzyme, catalyzes the Mg(2+)-dependent hydrolysis of ADP-ribose to AMP and ribose 5-phosphate. ADPR hydrolysis experiments conducted in the presence of H(2)(18)O and analyzed by electrospray mass spectrometry showed that the ADPRase-catalyzed reaction takes place through nucleophilic attack at the adenosyl phosphate. The structure of ADPRase in complex with Mg(2+) and a nonhydrolyzable ADPR analogue, alpha,beta-methylene ADP-ribose, reveals an active site water molecule poised for nucleophilic attack on the adenosyl phosphate. This water molecule is activated by two magnesium ions, and its oxygen contacts the target phosphorus (P-O distance of 3.0 A) and forms an angle of 177 degrees with the scissile bond, suggesting an associative mechanism. A third Mg(2+) ion bridges the two phosphates and could stabilize the negative charge of the leaving group, ribose 5-phosphate. The structure of the ternary complex also shows that loop L9 moves fully 10 A from its position in the free enzyme, forming a tighter turn and bringing Glu 162 to its catalytic position. These observations indicate that as part of the catalytic mechanism, the ADPRase cycles between an open (free enzyme) and a closed (substrate-metal complex) conformation. This cycling may be important in preventing nonspecific hydrolysis of other nucleotides.
Department of Biophysics and Biophysical Chemistry, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Sandra B
SB
Gabelli
Mario A
MA
Bianchet
Yuki
Y
Ohnishi
Yoshi
Y
Ichikawa
Maurice J
MJ
Bessman
L Mario
LM
Amzel
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D000096964:Nudix Hydrolases
D011755:Pyrophosphatases; D000096964:Nudix Hydrolases; C058796:ADP-ribose pyrophosphatase I