ROLE OF GLUTAMIC-ACID-988 OF HUMAN POLY-ADP-RIBOSE POLYMERASE IN POLYMER FORMATION - EVIDENCE FOR ACTIVE-SITE SIMILARITIES TO THE ADP-RIBOSYLATING TOXINS

Sequence similarities between the enzymatic region of poly-ADP-ribose polymerase and the corresponding region of mono-ADP-ribosylating bacte rial toxins suggest similarities in active site structure and catalyti c mechanism. Glu(988) Of the human polymerase aligns with the catalyti c glutamic acid of the toxins, and replacement of this residue with Gl n, Asp, or Ala caused major reductions in synthesis of enzyme-linked p oly-ADP-ribose. Replacement of any of 3 other nearby Glu residues had little effect. The Glu(988) mutations produced similar changes in acti vity in the carboxyl-terminal 40-kDa catalytic fragment fused to malto se binding protein: E988Q and E988A reduced polymer elongation > 2000- fold, and E988D similar to 20-fold. Smaller changes were seen in chain initiation. The mutations had little effect on the K-m of NAD, indica ting a predominantly catalytic function for Glu(988). The results supp ort the concept of similar active sites of the polymerase and the ADP- ribosylating toxins, Glu(988) may function in polymer elongation simil arly to the toxins' active site glutamate, as a general base to activa te the attacking nucleophile (in the case of the polymerase, the 2'-OH of the terminal adenosine group of a nascent poly-ADP-ribose chain).