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

Human ADP-ribosyltransferase 1 (<i>hs</i>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 <i>hs</i>ART1 activity has been shown to permit immune cell evasion in non-small cell lung cancer (NSCLC). Inhibition of <i>hs</i>ART1 decreases tumor efficacy and increases T-cell infiltration. <i>hs</i>ART1 is an emerging checkpoint target in select cancers. We performed the first kinetic characterization of the ADP-ribosyltransferase and NAD⁺ glycohydrolase activities of <i>hs</i>ART1. Without an l-arginine substrate, <i>hs</i>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 <i>hs</i>ART1 to l-arginine-like small molecule substrates gives over a 100-fold improvement in <i>k</i>_cat/<i>K</i>_m and <i>k</i>_cat relative to NAD⁺ hydrolysis. With ADP-ribose acceptors, <i>hs</i>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. <i>hs</i>ART1 is not diffusionally rate-limited on <i>k</i>_cat and only partially limited on <i>k</i>_cat/<i>K</i>_m for l-arginine methyl ester. The detailed description of the kinetic mechanism of <i>hs</i>ART1 can aid in the development of novel and selective inhibitors.