Mechanism of inhibition of poly(ADP-ribose) glycohydrolase by adenosine diphosphate (hydroxymethyl)pyrrolidinediol.

Abstract

Adenosine diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD), a nitrogen-in-the-ring analog of ADP-ribose, was recently shown to be a potent and specific inhibitor of poly(ADP-ribose) glycohydrolase. Analysis of the inhibition kinetics of the hydrolase by ADP-HPD using the method of Lineweaver and Burk yields a noncompetitive double-reciprocal plot. Both the intercept (1/V) versus [inhibitor] replot and the slope (Km/V) versus [inhibitor] replot are hyperbolic, indicating partial noncompetitive inhibition. Inhibitor dissociation constants Kii = 52 nM and Kis = 80 nM were determined for ADP-HPD by analysis of the intercept versus [inhibitor] and slope versus [inhibitor] replots. These results show that although ADP-HPD is extremely potent in inhibiting poly(ADP-ribose) glycohydrolase, its effectiveness is limited by its partial inhibition. ADP-HPD was significantly less potent as an inhibitor of the NAD glycohydrolase from Bungarus fasciatus venom. Analysis of the inhibition kinetics using the Lineweaver and Burk method indicated that ADP-HPD was a linear-competitive inhibitor of the NAD glycohydrolase with a Ki of 94 microM. The results indicate that at low concentration ADP-HPD will be a selective inhibitor of poly(ADP-ribose) glycohydrolase; however, complete inactivation of the activity will be difficult to obtain.