PURIFICATION AND PROPERTIES OF A PALMITOYL-PROTEIN THIOESTERASE THAT CLEAVES PALMITATE FROM H-RAS

H-Ras, the protein product of the cellular homologue of the Harvey ras oncogene, undergoes a complex series of post-translational modificati ons that include C-terminal isoprenylation, proteolysis, methylation, and palmitoylation. Palmitoylation has been shown to enhance the trans formation efficiency of H-Pas about 10-fold in vivo. A recent study (M agee, A. I., Gutierrez, L., McKay, I. A., Marshall, C. J., and Hall, A . (1987) EMBO J. 6, 3353-3357) has provided strong evidence that the p almitate undergoes a dynamic acylation-deacylation cycle, but details concerning the enzymology of this process and its regulation are lacki ng. To begin to dissect this event, we have developed an assay for the enzymatic removal of palmitate from [H-3]palmitate-labeled H-Ras. Thi s substrate was produced in a baculovirus expression system and was us ed to purify to homogeneity a novel 37-kDa enzyme from bovine brain cy tosol that removes the radiolabeled palmitate. The purified enzyme is sensitive to diethyl pyrocarbonate and insensitive to phenylmethyl-sul fonyl fluoride and N-ethylmaleimide. Interestingly, the thioesterase r ecognizes H-Ras as a substrate only when H-Ras is in its native confor mation (bound to Mg2+ and guanine nucleotide). The palmitoylated alpha subunits of the heterotrimeric G proteins are also substrates for the enzyme.