Differential Prenyl Pyrophosphate Binding to Mammalian Protein Geranylgeranyltransferase-I and Protein Farnesyltransferase and Its Consequence on the Specificity of Protein Prenylation

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 ␥ 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.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 (1-9). Two closely related enzymes protein farnesyltransferase (PFT) 1 and PGGT-I transfer prenyl groups from prenyl pyrophosphates to proteins that contain a C-terminal CaaX motif (C is cysteine, a is usually an aliphatic amino acid, and X is a variety of amino acids) (10 -13). The X residue of this motif plays a major role in recognition by these two enzymes (14). PFT preferentially transfers a farnesyl group to the cysteine residue of the CaaX motif when X is serine, methionine, glutamine, or cysteine, and possibly other residues. PGGT-I preferentially geranylgeranylates proteins having a C-terminal leucine or phenylalanine. PFT and PGGT-I consist of a common ␣ subunit and distinct ␤ subunits (15-18). A third enzyme, protein geranylgeranyltransferase-II, also known as Rab geranylgeranyltransferase, transfers the 20-carbon prenyl group to both cysteines of Rab proteins that have C-terminal sequences CXC, CC, or possibly CCXX (19,20). Although the C-terminal CaaX...

Protein geranylgeranyltransferase-I of Trypanosoma cruzi

“…Recombinant T. cruzi PFT and rat PGGT-I were produced in the baculovirus/Sf9 cell expression system and purified as described [8,16]. H-Ras-CVLL and other protein substrates produced in Escherichia coli were obtained as described [8].…”

“…H-Ras-CVLL and other protein substrates produced in Escherichia coli were obtained as described [8]. Biotinylated peptides were synthesized, and their structures were verified by mass-spectrometry as described [8].…”

“…H-Ras-CVLL and other protein substrates produced in Escherichia coli were obtained as described [8]. Biotinylated peptides were synthesized, and their structures were verified by mass-spectrometry as described [8]. The peptide segment VDWRKDDGVFMAERK of the T. cruzi PGGT-I β subunit sequence predicted from the cDNA was synthesized and used to raise the polyclonal antibodies in a rabbit (21 …”

“…After incubation at 30°C for 30 min, the reaction was terminated by adding 200 μl of 10% HCl in ethanol. The amount of radiolabeled protein product was quantified by the glass fiber filter method [8]. When a biotinylated peptide was used as a substrate, the reaction was terminated by boiling for 3 min, and 40 μl of avidin-agarose suspension (50% aqueous slurry, Pierce) was added to measure radioactivity transferred to the peptide as described [8].…”

“…Mammalian PGGT-I and PFT form a tight complex selectively with their substrates, geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP), respectively [7,8]. Structural and mechanistic studies revealed that critical residues involved in substrate binding occur in the β subunits of mammalian PFT and PGGT-I, and that PGGT-I discriminates FPP at the rate-determining product-release step in which GGPP displaces the geranylgeranylated product [9,10].…”