Skip to main content

Evaluation of an Alkyne-containing Analogue of Farnesyl Diphosphate as a Dual Substrate for Protein-prenyltransferases

The development of tools for proteomic analysis is an active area of research. Here, we report on the synthesis of 12-propargoxyfarnesyl diphosphate (1), an alkyne-containing analogue of farnesyl diphosphate (FPP), and its enzymatic incorporation into peptide substrates by both protein-farnesyltransferase (PFTase) and protein-geranylgeranyltransferase type I (PGGTase-I). Compound 1 was prepared from farnesol in 6 steps. Kinetic analyses indicate that 1 is incorporated into cognate peptide substrates by PFTase or PGGTase at concentrations and rates comparable to those of the natural lipid substrates for these enzymes, and mass spectrometric analyses proved the structures of the prenylated peptide products. Incubation of 1 in the presence of PFTase and PGGTase peptide substrates, and the cognate transferases, results in the simultaneous prenylation of both peptides emphasizing the dual substrate nature of 1. Thus, because 1 is a substrate for both enzymes, it can be used to introduce alkyne functionality into proteins that are normally either farnesylated or geranylgeranylated. This approach should be useful for a broad range of applications ranging from selective protein labeling to proteomic analysis.

This is a preview of subscription content, access via your institution.

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.
Fig. 8.

Abbreviations

DTT:

dithiothreitol

ESI-MS:

electrospray ionization mass spectrometry

HR:

high resolution

IU:

intensity units

FPP:

farnesyl diphosphate

GGPP:

geranylgeranyl diphosphate

HPLC:

high performance liquid chromatography

MS:

mass spectrometry

PFTase:

protein-farnesyltransferase

PGGTase:

protein-geranylgeranyltransferase

PPTs:

pyridinium p-toluenesulfonate

TFA:

trifluoroacetic acid

THF:

tetrahydrofuran

THP:

tetrahydropyranyl

TLC:

thin layer chromatography

References

Download references

Acknowledgments

The authors thank Dr. Tom Krick, Dr. Lee Ann Higgins, and Sean Murray in the Biological Mass Spectrometry Laboratory at the University of Minnesota for their assistance in obtaining the ESI-MS data reported here, and Dr. Daniel G. Mullen for his help with peptide synthesis. This work was supported by a grant from the National Institutes of Health (GM58442).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Mark D. Distefano.

Additional information

This paper is dedicated to the memory of Bruce Merrifield (1921–2006) for his pioneering development of solid-phase peptide synthesis, which has made possible myriad advances in chemical biology. For the present study, we used SPPS to prepare protein fragments that incorporate spectroscopic probes to reveal critical features in enzyme substrate recognition that have implications for human health.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hosokawa, A., Wollack, J.W., Zhang, Z. et al. Evaluation of an Alkyne-containing Analogue of Farnesyl Diphosphate as a Dual Substrate for Protein-prenyltransferases. Int J Pept Res Ther 13, 345–354 (2007). https://doi.org/10.1007/s10989-007-9090-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10989-007-9090-3

Keywords

  • alkyne
  • farnesyltransferase
  • geranylgeranyltransferase
  • peptide modification
  • prenylated peptide