Enzymatic incorporation of orthogonally reactive prenylazide groups into peptides using geranylazide diphosphate via protein farnesyltransferase: implications for selective protein labeling.

Protein farnesyltransferase (PFTase) catalyzes the attachment of a geranyl azide moiety to a peptide substrate, N-dansyl-Gly-Cys-Val-Ile-Ala-OH. The resulting azide-containing peptide was derivatized with a triphenylphosphine-based reagent to generate an O-alkyl imidate-linked product, rather than the amide-linked material expected via a Staudinger reaction. Since the CAAX box recognition motif (where the internal A residues are aliphatic amino acids) modified by PFTase can be incorporated into the C-terminus of virtually any polypeptide, this two-step procedure provides a general method for incorporating a diverse range of chemical modifications specifically near the C-terminus of proteins.

Use of synthetic isoprenoid analogues for understanding protein prenyltransferase mechanism and structure.

Protein prenylation involves the post-translational modification of specific protein-derived cysteine residues with farnesyl or geranylgeranyl groups through thioether linkages. Because a large number of proteins that participate in signal transduction processes require this modification, there has been intense interest in developing inhibitors of these enzymes and in clarifying the biological function of prenylation. Isoprenoid analogues have proven to be versatile tools for probing the mechanism and structure of prenyltransferases. Mechanistic probes have been created to investigate the stereochemical course and substituent effects in prenyltransferase catalyzed reactions. They have also been used to measure kinetic isotope effects and search for possible cationic intermediates. Photoaffinity labeling analogues containing either diazotrifluoropropionate or benzophenone units have been used to identify the location of isoprenoid binding sites in these enzymes. Biophysical probes incorporating fluorescent moieties or isotopic labels have been used to measure isoprenoid dissociation constants or prenyl group conformation when bound to the enzyme. Analogues containing noncognate alkene isomers or bulky substituents have also contributed to an understanding of isoprenoid recognition. Most recently, photoactive and isomeric isoprenylated analogues are providing insights into the function of protein prenylation.

Diazotrifluoropropionamido-containing prenylcysteines: syntheses and applications for studying isoprenoid-protein interactions.

Photoaffinity-labeled prenylcysteines (1 and 2) incorporating a diazotrifluoropropionamide-based photophore have been prepared. Photolyses of 2 in the presence of RhoGDI, a protein that interacts with prenylated proteins, and prenylcysteine-containing competitors demonstrate the effectiveness of this photoaffinity-labeled analogue as a tool for studying isoprenoid binding sites.