ADP‐ribosylargininyl Activity of Cholix Toxin Is Mediated Through Diffusible Intermediates

Abstract

OBJECTIVEWe discovered and characterized the ADP‐ribosylargininyl activity of the cholix toxin catalytic fragment, which was first identified as a diphthamide‐dependent ADP‐ribosyltransferase.METHODSWe utilized genetic approach to construct direct and reverse mutant enzymes and substrates in conjunction with biochemical assays including fluorescence/non‐fluorescence based enzymatic assays, protein/peptide‐2D‐electrophoresis analysis.RESULTSOur studies show that the transfer of ADP‐ribose to toxin itself takes place by a predominantly intramolecular mechanism. Multiple arginine residues, located in the proximity of the catalytic site but also at distal sites, can be the ADP‐ribose acceptor in the auto‐reaction. Kinetic studies of a model enzyme, M8, showed that a diffusible intermediate preferentially modifies arginine residues in proximity to the NAD+ binding pocket. ADP‐ribosylarginine activity of cholix toxin catalytic fragment could also modify exogenous substrates, such as oligo arginine peptides and eEF2(H715R) mutant. Auto‐ADP‐ribosylation of cholix toxin appears to have negative regulatory effect on its enzymatic activities. However, at the presence of both endogenous and exogenous substrates, the ADP‐ribosylation of exogenous substrate occurred more efficiently than endogenous substrate.CONCLUSIONSThe lifetime of the hypothetical intermediate exceeds recorded and predicted lifetime for the cognate oxocarbenium ion. We therefore hypothesize a strained form of NAD+ intermediate generated through an error‐prone mechanism of the enzyme to react with arginine residues in a proximity dependent manner.