Kinetic Mechanism of the Emergent Anticancer Target, Human ADP-ribosyltransferase 1.

Human ADP-ribosyltransferase 1 (ART1, EC: 2.4.2.31) is a membrane-associated GPI-anchored, arginine-specific, mono-ADP-ribosyltransferase. The enzyme resides on the endoplasmic reticulum and extracellular cell surface, where it catalyzes the transfer of ADP-ribose (ADPR) from NAD to arginine residues of neighboring target proteins, forming free nicotinamide (NAM) and N-linked mono-ADP-ribosylation (MARylation) of the target protein. Arginine-specific MARylation regulates the target's function and cellular roles. Dysregulation of ART1 activity has been shown to permit immune cell evasion in non-small cell lung cancer (NSCLC). Inhibition of ART1 decreases tumor efficacy and increases T-cell infiltration. ART1 is an emerging checkpoint target in select cancers. We performed the first kinetic characterization of the ADP-ribosyltransferase and NAD glycohydrolase activities of ART1. Without an l-arginine substrate, ART1 slowly hydrolyses NAD into NAM and ADPR through an ordered kinetic mechanism. NAD binding and hydrolysis are followed by the ordered release of NAM followed by ADPR. The ADP-ribosyltransferase activity of ART1 to l-arginine-like small molecule substrates gives over a 100-fold improvement in / and relative to NAD hydrolysis. With ADP-ribose acceptors, ART1 proceeds through a partially ordered mechanism, whereby the substrate binding of NAD and l-arginine-like substrate is random. Chemistry proceeds through a ternary complex, and product release is ordered, with NAM first, followed by the ADP-ribosylated acceptor. ART1 is not diffusionally rate-limited on and only partially limited on / for l-arginine methyl ester. The detailed description of the kinetic mechanism of ART1 can aid in the development of novel and selective inhibitors.