Abstract
Protein geranylgeranyltransferase-I (PGGT-I) and protein farnesyltransferase (PFT) attach geranylgeranyl and farnesyl groups, respectively, to the C termini of eukaryotic cell proteins. In vitro, PGGT-I and PFT can transfer both geranylgeranyl and farnesyl groups from geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP) to their protein or peptide prenyl acceptor substrates. In the present study it is shown that PGGT-I binds GGPP 330-fold tighter than FPP and that PFT binds FPP 15-fold tighter than GGPP. Therefore, in vivo, where both GGPP and FPP compete for the binding to prenyltransferases, PGGT-I and PFT will likely be bound predominantly to GGPP and FPP, respectively. Previous studies have shown that K-Ras4B and the Ras-related GTPase TC21 are substrates for both PGGT-I and PFT in vitro. It is shown that TC21 can compete with the C-terminal peptide of the gamma subunit of heterotrimeric G proteins and with the C-terminal peptide of lamin B for geranylgeranylation by PGGT-I and for farnesylation by PFT, respectively. K-Ras4B competes in both cases but is almost exclusively farnesylated by PFT in the presence of the lamin B peptide competitor. Rapid and single turnover kinetic studies indicate that the rate constant for the PGGT-I-catalyzed geranylgeranyl transfer step of the reaction cycle is 14-fold larger than the steady-state turnover number, which indicates that the rate of the overall reaction is limited by a step subsequent to prenyl transfer such as release of products from the enzyme. PGGT-I-catalyzed farnesylation is 37-fold slower than geranylgeranylation and is limited by the farnesyl transfer step. These results together with earlier studies provide a paradigm for the substrate specificity of PGGT-I and PFT and provide information that is critical for the design of prenyltransferase inhibitors as anti-cancer agents.
Highlights
Modification of the C termini of specific eukaryotic proteins by attachment of either 15-carbon farnesyl or 20-carbon geranylgeranyl groups is required for their proper membrane targeting and functional activation [1,2,3,4,5,6,7,8,9]
Recent studies show that photoaffinity analogs of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) exclusively label the  subunits of Protein geranylgeranyltransferase-I (PGGT-I) and protein farnesyltransferase (PFT) [26, 31, 32], and cross-linking studies with photoreactive peptide prenyl acceptors suggest that prenyl acceptors bind to an ␣ subunit/ subunit interface [33]
Differential Binding of GGPP and FPP to PGGT-I and PFT—We have previously shown with in vitro reactions that purified bovine PGGT-I is able to transfer both geranylgeranyl
Summary
Materials—Biotin-␥6-CAIL and biotin-lamin B-CAIS were synthesized and analyzed by mass spectroscopy as described previously [34]. [3H]FPP and [3H]GGPP (15 Ci/mmol, labeled on carbon-1) and unlabeled FPP and GGPP were purchased from American Radiolabeled Chemicals. PGGT-I and PFT Assays—To assay the geranylgeranylation activity of PGGT-I, 1 M [3H]GGPP (15 Ci/mmol) and 5 M biotin-␥6-CAIL were incubated with 0.1 g of rat PGGT-I in a total volume of 20 l containing 30 mM potassium phosphate, 5 mM DTT, 0.5 mM MgCl2, 20 M ZnCl2, pH 7.7. The standard assay for PFT was carried out with 10 M H-Ras-CVLS and 3.3 M [3H]FPP in a total volume of 20 l containing 30 mM potassium phosphate, 5 mM DTT, 10 mM MgCl2, 20 M ZnCl2, 25 mM NaCl, pH 7.7. The column was spun for 2 min at 2,500 rpm in an HB-4 swinging-bucket rotor and washed once with 50 l of the same buffer by spinning in the same way These two eluants that contain protein but negligible amounts of unbound prenyl pyrophosphate were combined, and the mixture was submitted to scintillation counting. Counting efficiency was determined by counting a known amount of [3H]GGPP together with blank silica gel
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