Protein farnesyl transferase target selectivity is dependent upon peptide stimulated product release.
Protein farnesyl transferase (FTase) catalyzes transfer of a 15 carbon farnesyl lipid to cysteine in the C-terminal Ca1a2X sequence of numerous proteins including Ras. Previous studies have shown that product release is rate limiting and is dependent on binding of either a new peptide or isoprenoid diphosphate substrate. While considerable progress has been made in understanding how FTase distinguishes between related target proteins, the relative importance of the two pathways for product release on substrate selectivity is unclear. A detailed analysis of substrate stimulated product release has now been performed and provides new insights into the mechanism of FTase target selectivity. To clarify how FTase selects between different Ca1a2X sequences, we have examined the competition of various peptide substrates for modification with the isoprenoids farnesyl diphosphate (FPP) and anilinogeranyl diphosphate (AGPP). We find that reactivity of some competing peptides is correlated with apparent Kmpeptide, while the reactivity of others is predicted by the selectivity factor apparent kcat/Kmpeptide. The peptide target selectivity also depends on the structure of the isoprenoid donor. Additionally, we observe two peptide substrate concentration dependent maxima and substrate inhibition in the steady-state reaction which require a minimum of three peptide binding states for the steady-state FTase reaction mechanism. We propose a model for the FTase reaction mechanism that, in addition to FPP stimulated product release, incorporates peptide binding to the FTase-FPP complex and the formation of an FTase-product-peptide complex followed by product release leading to an inhibitory FTase-peptide complex as a natural consequence of catalysis to explain these results.
Troutman
Jerry M
JM
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0084, USA.
Andres
Douglas A
DA
Spielmann
H Peter
HP
D000814:Aniline Compounds / Q000737:chemistry / Q000378:metabolism; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D051231:Farnesyltranstransferase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
C412022:8-anilinogeranyl pyrophosphate; D000814:Aniline Compounds; D011756:Diphosphates; D010455:Peptides; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D051231:Farnesyltranstransferase