Lh. Meng et al., THE ANTIPROLIFERATIVE AGENT DIDEMNIN-B UNCOMPETITIVELY INHIBITS PALMITOYL PROTEIN THIOESTERASE, Biochemistry, 37(29), 1998, pp. 10488-10492
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.