Protein farnesyltransferase (FTase), a heterodimer enzyme consisting of alpha and beta subunits, catalyzes the addition of farnesyl groups to 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 that of a closely related enzyme, geranylgeranyltransferase type I (GGTase 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 mutants can function as either FTase or GGTase I beta subunit in vivo, although some differences in efficiency were observed. These results point to the importance of two distinct regions (one at 159 and the other 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 asparagine 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 beta. 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 Y362L mutant proteins showed that their kcat/Km values for GGTase I substrate are increased about 20-fold compared with that of the wild type protein. These results demonstrate that the conversion of the protein substrate specificity of FTase to that of GGTase I can be accomplished by introducing a distinct size amino acid at either of the two residues, 159 and 362.

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Journal Article | Research Support, U.S. Gov't, P.H.S.

Authors

Del Villar K, Mitsuzawa H, Yang W, Sattler I, Tamanoi F

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