Transition-state analysis of the arginine-specific human ADP-ribosyltransferase 1.

The ADP-ribosylation of proteins is a versatile, reversible, posttranslational modification involved in the dynamic regulation of numerous cellular processes. Human ADP-ribosyltransferase 1 (hsART1, EC: 2.4.2.31) is a membrane-associated, GPI-anchored, mono-ADP-ribosyltransferase selective for mono-ADP ribosylation (MARylation) of L-arginine residues. Dysregulation of hsART1 activity has been shown to permit immune cell evasion in non-small cell lung cancer (NSCLC) through elevated MARylation at Arg125 of the purinergic type 2 receptor (P2X7) in P2X7-positive T cells, resulting in NAD+-induced cell death (NICD) of tumor-penetrating immune cells. With hsART1 emerging as an immunotherapy target in select cancers, there is a need to develop small-molecule inhibitors. The transition state (TS) for the MARylation of P2X7 peptide was determined from kinetic isotope effect (KIE) measurements of 3H-, 14C-, 18O-, and 15N-labeled NAD+ substrates. Quantum mechanical (QM) calculations of the reaction coordinate, mapped with experimental KIEs, identify a TS geometry consistent with a highly dissociative, asymmetric, concerted mechanism with minimal contributions from the leaving group nicotinamide (NAM) and minor contributions from the incoming L-arginine guanidinium. The absence of a normal deuterium solvent isotope effect identifies a positively charged guanidinium nucleophile, leading to a dication N-ribosyltransferase mechanism. Together with the unusual, normal 18O-O4' KIE, we identified unique charge accumulation across the oxocarbenium at the TS caused by an increased bond order between the C1'-C2', and decreased bond order between the C4'-O4' of the nicotinamide mononucleotide ribose. This is the first L-arginine-specific ADP-ribosylation TS to be characterized, a step toward the design of TS analogs.