Farnesyl Diphosphate Analogues with Aryl Moieties Are Efficient Alternate Substrates for Protein Farnesyltransferase

Abstract

Farnesylation is an important post-translational modification essential for proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of analogue transfer to peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple turnover activity depends on the analogue structure. Analogues where the first isoprene is replaced by a benzyl group and an analogue where each isoprene is replaced by an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single turnover rate constant for alkylation does depend on the Ca1a2X peptide sequence. These results suggest that the isoprenoid transition state conformation is preferred over the inactive E•FPP• Ca1a2X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for design of novel inhibitors.

Rapid Analysis of Protein Farnesyltransferase Substrate Specificity Using Peptide Libraries and Isoprenoid Diphosphate Analogues

“…Similarly, isoprenoid analogues are being developed to disrupt interactions with prenylated proteins. 57 In those cases, it will be important to know whether such analogues are incorporated into prenylated proteins with a specificity profile similar to farnesyl groups. While in vitro assays with individual peptides do allow the catalytic efficiencies of different isoprenoid substrates to be compared, such experiments do not allow global variations in peptide substrate efficiency to be studied.…”

Protein Prenylation: Enzymes, Therapeutics, and Biotechnology Applications

“…For example, analog 13 , where all isoprene units were replaced with aryl groups, was an efficient FTase substrate. 69 They reported that the anilinogeranyl-based isoprenoid analogs 14 and 15 , which have 2–5 orders of magnitude less hydrophobicity compared with FPP, were substrates for FTase. Proteins modified with these analogs were processed by downstream enzymes Rce1 and Icmt; however, the resulting modified proteins were not biologically active, indicating the importance of increased hydrophobicity upon prenylation.…”

Efficient farnesylation of an extended C-terminal C(x)3X sequence motif expands the scope of the prenylated proteome

“…, Km = 1.5 µM), (65) kcat values for both dns-GC(x)3X peptides are reduced ~30-fold while Km increases ~4-fold for dns-GCAVGP and decreases ~3-fold for dns-GCMIIM. When compared to dns-GCMII and dns-GCAVG, the potential sequences derived from proteolytic trimming, kcat/Km was increased 25-fold for dns-GCAVG relative to dns-GCVAGP and decreased 2-fold for dns-GCMII relative to dns-GCMIIM.…”