High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase beta-subunit C-terminal mutants

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimeric enzyme that catalyzes post-translational farnesylation of many cellular regulatory prot eins including p21 Pas. To facilitate the construction of site-directed mut ants, a novel translationally coupled, two-cistron Escherichia coli express ion system for rat FPT has been developed. This expression system enabled y ields of >5 mg of purified protein per liter of E.coli culture to be obtain ed. The E. coli-derived FPT demonstrated an activity comparable to that of protein isolated from other sources. The reported expression system was use d to construct three beta-subunit C-terminal truncation mutants, Delta 5, D elta 10 and Delta 14, which were designed to eliminate a lattice interactio n between the beta-subunit C-terminus of one molecule and the active site o f a symmetry-related molecule. Steady-state kinetic analyses of these mutan ts showed that deletion up to 14 residues at the C-terminus did not reduce the value of k(cat); however, K(m)values for both peptide and FPP increased 2-3-fold, A new crystalline form of FPT was obtained for the Delta 10 C-te rminal mutant grown in the presence of the substrate analogs acetyl-Cys-Val -Ile-Met-COOH peptide and alpha-hydroxyfarnesylphosphonic acid. The crystal s diffract to beyond 2.0 Angstrom resolution. The refined structure clearly shows that both substrate analogs adopt extended conformations within the FPT active site cavity.