Identification of an inhibitor binding site of poly(ADP-ribose) glycohydrolase.
Polymers of ADP-ribose involved in the maintenance of genomic integrity are converted to free ADP-ribose by the action of poly(ADP-ribose) glycohydrolase (PARG). As an approach to mapping functions of PARG onto the amino acid sequence of the protein, we report here experiments that identify an amino acid residue involved in the binding of potent PARG inhibitors. A photoreactive inhibitor, [alpha-(32)P]-8-azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol (8-N(3)-ADP-HPD), was used to photolabel a recombinant bovine PARG catalytic fragment (rPARG-CF). N-Terminal sequencing of tryptic and subtilitic peptides of photoderivatized rPARG-CF identified tyrosine 796 (Y796), a residue conserved in PARG across a wide range of organisms, as a site of photoderivatization. Site-directed mutants where this tyrosine residue was replaced with an alanine residue (Y796A) had a nearly 8-fold decrease in catalytic efficiency (k(cat)/K(M)), while replacement with a tryptophan residue (Y796W) had little effect on catalytic efficiency. Surface plasmon resonance spectroscopy using the PARG inhibitor 8-(aminohexyl)amino-ADP-HPD demonstrated that the binding constant of the inhibitor for Y796A was 21-fold lower (K(D) = 170 nM) than that of wild-type PARG (K(D) = 8.2 nM), while Y796W displayed a binding affinity similar to that of the wild-type enzyme. Our results indicate that Y796 is involved in inhibitor binding to PARG via a ring stacking interaction and identify a highly conserved region of the protein that putatively contains other residues involved in catalytic activity and/or substrate recognition.
Koh
David W
DW
College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, USA.
Patel
Chandra N
CN
Ramsinghani
Sushma
S
Slama
James T
JT
Oliveira
Marcos A
MA
Jacobson
Myron K
MK
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005091:Exons; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D014443:Tyrosine; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase