THE ANTIPROLIFERATIVE AGENT DIDEMNIN-B UNCOMPETITIVELY INHIBITS PALMITOYL PROTEIN THIOESTERASE

Dynamic protein palmitoylation has been proposed to regulate GTP-bindi ng proteins by controlling their membrane association and thus their a ccess to key signaling proteins. While the palmitoyl protein thioester ase(s) responsible for depalmitoylation of plasma membrane-associated signaling proteins has (have) not been identified, the lysosomal palmi toyl protein thioesterase 1 (PPT1) has proven useful in in vitro studi es of membrane localization requirements of GTP-binding proteins. We h ave previously reported the binding of the antiproliferative cyclic de psipeptide didemnin B to PPT1. To investigate the nature of this bindi ng and its possible effects on PPT1 enzymatic activity, human PPT1 was expressed in an insect cell baculoviral system, and inhibition assays were performed using both [H-3]paimitoyl Ha-Ras and myristoyl-Coil as PPT1 substrates. Didemnin B was shown to inhibit recombinant human PP T1 with a K-i of 92 nM. Kinetic analysis of this inhibition revealed t hat didemnin B inhibits PPT1 uncompetitively. Providing biochemical su pport for an uncompetitive mode of inhibition, in vitro binding studie s of PPT1 and didemnin indicate that the natural product binds prefere ntially to the enzyme-substrate complex PPT1-palmitoyl-CoA. As the fir st described inhibitor of PPT1, didemnin B may prove to be a useful to ol in the investigation of protein palmitoylation regulation.