CHARACTERIZATION OF RECOMBINANT HUMAN FARNESYL-PROTEIN TRANSFERASE - CLONING, EXPRESSION, FARNESYL DIPHOSPHATE BINDING, AND FUNCTIONAL HOMOLOGY WITH YEAST PRENYL-PROTEIN TRANSFERASES

We have isolated cDNAs encoding the alpha and beta subunits of human f arnesyl-protein transferase (FPTase). The proteins encoded by these tw o cDNAs are 93-95% identical to the corresponding subunits of bovine a nd rat FPTase and show regions of homology with proteins encoded by Sa ccharomyces cerevisiae prenyl-protein transferase genes. Human FPTase expressed in Escherichia coli from a translationally coupled operon ha d kinetic properties similar to those of FPTase isolated from bovine b rain. Examination of farnesyl diphosphate binding indicated that while neither individual subunit was capable of isoprenoid binding, a radio labeled farnesyl diphosphate analog could be specifically photo-cross- linked to the beta subunit of FPTase holoenzyme. To further analyze su bunit structure-function and to detect functional similarities with ye ast prenyl-protein transferases (FPTase and two geranylgeranyl-protein transferases), amino acid changes homologous to those found in mutant yeast prenyl-protein transferase subunits were made in the subunits o f human FPTase. Substitutions in either the alpha or beta subunits tha t decrease the activity of yeast prenyl-protein transferases were also observed to impair human FPTase. Kinetic analyses showed that these m utant human FPTases have K(m) and k(cat) values that are altered with respect to wild-type human FPTase.