Structure
ADPRase is a dimer of two identical monomers, each of which contain 209 amino acids. The two monomers are folded into two distinct structural domains and with two equivalent catalytic sites. The C-terminal domain consists of the Nudix sequence mentioned above and the N-terminal domain is primarily involved in dimer stabilization. As noted earlier, the Nudix fold is the catalytic part of the enzyme, but it should also be noted that both domains are involved in the active site and they both help with the attachment and coordination of H2O, Mg2+, and the ADP-ribose substrate.
The picture to the right shows the active site of ADPRase in complex with AMPCPR and Mg2+. AMPCPR is a nonhydrolyzable analog of ADP-ribose, and thus functions as an inhibitor of ADPRase. The picture below reveals the first and second Mg2+ ions, which serve to coordinate the attacking water molecule so that it is perfectly in line with the scissile bond (O-P-O bond is 177 degrees) and poised for attack. A glutamate side chain (E162) will deprotonate the water molecule, and then the hydroxide ion will attack nucelophillicly. This occurs when the water molecule is 3.0 angstroms away from the phosphorus molecule it is attacking. By using H2O18, it has been found that the water molecule attacks the adenosyl phosphate. This evidence is a refutation of a previous study that suggested that hydrolysis of ADP-ribose could proceed by nucleophilic attack at either alpha- or beta- phosphate (Gabelli, et al. 2001). After the water molecule attacks, an intermediate is formed, and then ribose 5-phosphate is kicked off as a leaving group. The third active site Mg2+ ion is used to stabilize the negative charge of the phosphate group as it leaves, and is thus situated perfectly between the two phosphates.
When the substrate is bound, the structure of the enzyme differs in shape. With the substrate bound, Loop L9 moves 10 angstroms from its original position in the free enzyme, which forms a tighter turn and brings E162 to its catalytic position, meaning this enzyme cycles between an open (free enzyme) and a closed (substrate-metal complex) conformation. This is important because it prevents nonspecific hydrolysis of other nucleotides. ADPRase is highly specific for ADPR, as it has been shown that its Km for ADPR is lower by at least two orders of magnitude than for other sugar nucleotides.
Read more about this topic: ADP-ribose Diphosphatase
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