Monitoring the three enzymatic activities involved in posttranslational modifications of Ras proteins

Ras proteins are important oncogenes that are involved in more than 30% of human cancers. To become functional, Ras proteins have to undergo at least three posttranslational modifications: farnesylation, endoproteolysis, and carboxyl-methylation. The enzymes, which catalyze the three reactions, are under investigation as potential anti-cancer targets. Here we developed a highly sensitive capillary electrophoresis (CE) method for monitoring these three enzymatic activities. In this method, a fluorescently labeled pentapeptide is used as a substrate. The substrate is sequentially converted to three products by the enzymes: protein farnesyltransferase, endoprotease, and methyltransferase. The three products retain the fluorescent label. The substrate and the three products are separated by CE and detected by laser-induced fluorescence. The method is characterized by: (i) the efficiency of greater than 400,000 theoretical plates, (ii) the resolution of greater than 7, and (iii) the limit of detection of as low as 800 molecules of the enzymatic product. Using the new method we measured kinetic parameters of endoprotease-catalyzed cleavage of three carboxy-terminal amino acids from the farnesylated substrate: V-max = 7 nmol min(-1) mg(-1) of the protein and K-m = 1.3 muM. The method will find its applications in studies of inhibitors of the three enzymes in search for new Ras-targeting anti-cancer agents. (C) 2004 Elsevier B.V. All rights reserved.