Role of Glutamic Acid 988 of Human Poly-ADP-ribose Polymerase in Polymer Formation

Abstract

Sequence similarities between the enzymatic region of poly-ADP-ribose polymerase and the corresponding region of mono-ADP-ribosylating bacterial toxins suggest similarities in active site structure and catalytic mechanism. Glu988 of the human polymerase aligns with the catalytic glutamic acid of the toxins, and replacement of this residue with Gln, Asp, or Ala caused major reductions in synthesis of enzyme-linked poly-ADP-ribose. Replacement of any of 3 other nearby Glu residues had little effect. The Glu988 mutations produced similar changes in activity in the carboxyl-terminal 40-kDa catalytic fragment fused to maltose-binding protein: E988Q and E988A reduced polymer elongation > 2000-fold, and E988D approximately 20-fold. Smaller changes were seen in chain initiation. The mutations had little effect on the Km of NAD, indicating a predominantly catalytic function for Glu988. The results support the concept of similar active sites of the polymerase and the ADP-ribosylating toxins. Glu988 may function in polymer elongation similarly to the toxins' active site glutamate, as a general base to activate the attacking nucleophile (in the case of the polymerase, the 2'-OH of the terminal adenosine group of a nascent poly-ADP-ribose chain).