AMINO-ACID SUBSTITUTIONS THAT CONVERT THE PROTEIN SUBSTRATE-SPECIFICITY OF FARNESYLTRANSFERASE TO THAT OF GERANYLGERANYLTRANSFERASE TYPE-I

Protein farnesyltransferase (FTase), a heterodimer enzyme consisting o f alpha and beta subunits, catalyzes the addition of farnesyl groups t o the C termini of proteins such as Ras, In this paper, we report that the protein substrate specificity of yeast FTase can be switched to t hat of a closely related enzyme, geranylgeranyltransferase type I (GGT ase I) by a single amino acid change at one of the three residues: Ser -159, Tyr-362, or Tyr-366 of its beta-subunit, Dpr1. All three Dpr1 mu tants can function as either FTase or GGTase I beta subunit in vivo, a lthough some differences in efficiency were observed, These results po int to the importance of two distinct regions (one at 159 and the othe r at 362 and 366) of Dpr1 for the recognition of the protein substrate , Analysis of the protein, after site directed mutagenesis was used to change Ser-159 to all possible amino acids, showed that either aspara gine or aspartic acid at this position allowed FTase beta to function as GGTase I beta. A similar site directed mutagenesis study on Tyr-362 showed that leucine, methionine, or isoleucine at this position also resulted in the ability of mutant FTase beta to function as GGTase I b eta, Interestingly, in both position 159 and 362 substitutions, amino acids that could change the protein substrate specificity had similar van der Waals volumes. Biochemical characterization of the S159N and Y 362L mutant proteins showed that their k(cat)/K-m values for GGTase I substrate are increased about 20-fold compared with that of the wild t ype protein, These results demonstrate that the conversion of the prot ein substrate specificity of FTase to that of GGTase I can be accompli shed by introducing a distinct size amino acid at either of the two re sidues, 159 and 362.