Structure-function analysis of residues important in zinc binding and activity in protein farnes yltransferase

Protein farnesyltransferase (FTase) is a zinc metalloenzyme that performs a post-translational modification on many proteins that is critical for their function. The importance of cysteine residues in FTase activity was investigated using thiol-specific reagents. Through the use of selective chemical modification by [H3]NEM in conjunction with peptide sequencing and analysis by site-directed mutagenesis, Cys299 in the β subunit of FTase was identified as an essential cysteine residue that played a critical role in catalysis. We hypothesized that it was one of the residues that directly coordinate the zinc atom in the enzyme. This hypothesis has just been confirmed by direct structure analysis. To further explore the role of zinc in catalytic mechanism of FTase, mutasenesis on the remaining ligands of the metal is being performed. His in the βsubunit has been changed to Ala, Gin or Glu by sitedirected mutagenesis. Although the abilities to bind FPP by all these mutant FTases were similar to that by the wild-type enzyme, their residual activities were quite different. Most interestingly, the H362E mutant retained about 8 % the activity of wild-type FTase, and a preliminary kinetic analysis of this mutant enzyme indicates that the rate-limiting step has changed from product release (in the wild-type enzyme) to the chemical step of actual product formation. Additional kinetic analyses of this mutant and others being prepared should yield important information on participation of zinc in the catalytic mechanism of FTase.

Duke Scholars

Author Patrick John Casey Pharmacology & Cancer Biology