Functional characterization of the RAS processing enzyme Rce1

Proteins containing a C-terminal CaaX motif undergo post-translational modifications required for their localization to membrane surfaces and biologic functions. These post-translational modifications consist of: lipidation by a protein prenyltranferase, endoproteolytic cleavage by RAS-converting enzyme 1 (Rce1), and carboxyesterification by isoprenylcysteine carboxymethyltransferase (ICMT). These enzymatic processes are possible targets for cancer therapeutics, as they are necessary for the signaling function of RAS GTPases and other oncoproteins. However, difficulties associated with the biochemical purification and characterization of membrane embedded proteins have limited our understanding of the enzymatic mechanism of Rce1, which resides within the ER membrane. To address this, we identified a biochemically tractable ortholog of Rce1 from Drosophila melanogaster and developed a strategy to purify the enzyme in its catalytically active form. Purified Rce1 recapitulates known enzymatic properties, including a requirement for its substrates to contain an attached prenyl lipid. Characterization of the enzyme's kinetic properties reveal slow enzymatic turnover (~ 20 hr -1 ), which is consistent with other integral membrane proteases. We find that Zn 2+ and the lipid PA inhibit Rce1, raising the possibility these factors may regulate its catalytic activity in vivo . Mass spectrometry analysis of the purified protein indicates that the enzymatic mechanism does not utilize metal ions. We hypothesize that conserved histidine and glutamate amino acids have catalytic roles, which differentiates Rce1 from most other proteases. This study provides insights into the catalytic mechanism of the enzyme, which could be useful for developing mechanism-based inhibitors.