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 proteins including p21 Ras. To facilitate the construction of site-directed mutants, a novel translationally coupled, two-cistron Escherichia coli expression system for rat FPT has been developed. This expression system enabled yields of >5 mg of purified protein per liter of E.coli culture to be obtained. The E.coli-derived FPT demonstrated an activity comparable to that of protein isolated from other sources. The reported expression system was used to construct three beta-subunit C-terminal truncation mutants, Delta5, Delta10 and Delta14, which were designed to eliminate a lattice interaction between the beta-subunit C-terminus of one molecule and the active site of a symmetry-related molecule. Steady-state kinetic analyses of these mutants showed that deletion up to 14 residues at the C-terminus did not reduce the value of kcat; however, Km values for both peptide and FPP increased 2-3-fold. A new crystalline form of FPT was obtained for the Delta10 C-terminal mutant grown in the presence of the substrate analogs acetyl-Cys-Val-Ile-Met-COOH peptide and alpha-hydroxyfarnesylphosphonic acid. The crystals diffract to beyond 2.0 A resolution. The refined structure clearly shows that both substrate analogs adopt extended conformations within the FPT active site cavity.