Catalytic properties of the integral membrane RAS protease Rce1

The integral membrane protease Rce1 cleaves prenylated CaaX proteins, including all RAS GTPases, as part of their post-translational maturation. This cleavage, which removes the -aaX residues, is necessary for the proper cellular location and signaling properties of RAS and its oncogenic forms. Poor biochemical stabilities of yeast and mammalian Rce1 have hindered the purification of Rce1 in active form and limited studies addressing its catalytic properties. Here we identify that Drosophila melanogaster Rce1 has good biochemical stability and is catalytically active when purified. The purified protein recapitulates known properties of Rce1, including specificity for prenylated CaaX substrates. Like other integral membrane proteases, the enzyme has a low turnover number (k_cat), of approximately 20 hr^-1. Pre-steady-state kinetic analysis reveals that product release is the rate limiting step. Our data indicate that Rce1 is inhibited by nanomolar levels of Zn²⁺, but that metals are not involved in catalysis. The data suggest that Zn²⁺ is a competitive inhibitor with respect to the substrate. The signaling lipid PA also inhibits the enzyme in vitro, raising the possibility that the cellular activity of Rce1 may be modulated by lipids and/or Zn²⁺. These studies combined with structural predictions and prior investigation lead to the hypothesis that the active site contains a catalytic Glu-His-His triad of amino acids that coordinate the substrate and form the inhibitory Zn²⁺ site. This work enhances the understanding of the enzyme and gives insight into means for Rce1 inhibition.