title: ADP-Ribosylargininyl reaction of cholix toxin is mediated through diffusible intermediates.
abstract: BACKGROUND
  Cholix toxin is an ADP-ribosyltransferase found in non-O1/non-O139 strains of Vibrio cholera. The catalytic fragment of cholix toxin was characterized as a diphthamide dependent ADP-ribosyltransferase.
  
  
  RESULTS
  Our studies on the enzymatic activity of cholix toxin catalytic fragment show that the transfer of ADP-ribose to toxin takes place by a predominantly intramolecular mechanism and results in the preferential alkylation of arginine residues proximal to the NAD+ binding pocket. Multiple arginine residues, located near the catalytic site and 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 reacted with arginine residues in proximity to the NAD+ binding pocket. ADP-ribosylarginine activity of cholix toxin catalytic fragment could also modify exogenous substrates. Auto-ADP-ribosylation of cholix toxin appears to have negatively regulatory effect on ADP-ribosylation of exogenous substrate. However, at the presence of both endogenous and exogenous substrates, ADP-ribosylation of exogenous substrates occurred more efficiently than that of endogenous substrates.
  
  
  CONCLUSIONS
  We discovered an ADP-ribosylargininyl activity of cholix toxin catalytic fragment from our studies in auto-ADP-ribosylation, which is mediated through diffusible intermediates. The lifetime of the hypothetical intermediate exceeds recorded and predicted lifetimes for the cognate oxocarbenium ion. Therefore, a diffusible strained form of NAD+ intermediate was proposed to react with arginine residues in a proximity dependent manner.
authors.affiliation: Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston 02114, MA, USA. mvsung@gmail.com.
authors.forename: Vicky M-H
authors.initials: VM
authors.lastname: Sung
authors.forename: Chia-Lun
authors.initials: CL
authors.lastname: Tsai
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000633:toxicity; D002772:Cholera Toxin / Q000378:metabolism*; D004058:Diffusion
chemical_list: D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; C043357:ADP-ribosylarginine