In an effort to create photoactive analogues of FPP with enhanced stability, two new compounds have been prepared. Probe 3 contains an amide-linked DATFP moiety, is an alternative substrate for ScPFTase and covalently modifies a number of prenyltransferases upon photolysis. However, 3 rearranges to a photochemically inactive triazolone limiting its potential utility. Probe 4 employs an alkyl ester in lieu of the allylic ester found in 2; that substitution renders the linkage between the DATFP group and the isoprenoid significantly more stable to acidic and basic conditions. Compound 4 is an alternative substrate for ScPFTase. Computer assisted docking and crystallographic analysis indicate that 4 binds to RnPFTase in a manner similar (although not identical) to the natural substrate FPP; those suggest that 4 is a reasonable surrogate for a farnesyl group. Photolysis of 4 in the presence of a several different types of FPP-utilizing enzymes results in crosslinking; of particular note, photolysis of 4 in crude E. coli extract expressing ScPFTase results in selective labeling of that enzyme even though it is present as approximately 1% of the total protein. We are currently employing 4 in experiments designed to identify proteins involved in latex and sesquiterpene biosynthesis. Finally, the ability to introduce 4 into peptides, established here, suggests that it should be possible to prepare peptides and proteins that incorporate photoactive isoprenoids that can be used to study the processing of prenylated proteins.(, , -) Given our recent development of cell penetrating prenylated peptides, it should be possible to perform these experiments in living cells.() Efforts to accomplish this are currently under way.
Protein prenylation is a posttranslational lipid modification in which C15 and C20 isoprenoid units are linked to specific protein-derived cysteine residues through a thioether linkage. This process is catalyzed by a class of enzymes called prenyltransferases that are being intensively studied due to the finding that Ras protein is farnesylated coupled with the observation that mutant forms of Ras are implicated in a variety of human cancers. Inhibition of this posttranslational modification may serve as a possible cancer chemotherapy. Here, the syntheses of two new farnesyl diphosphate (FPP) analogues containing photoactive benzophenone groups are described. Each of these compounds was prepared in six steps from dimethylallyl alcohol. Substrate studies, inhibition kinetics, photoinactivation studies, and photolabeling experiments are also included; these experiments were performed with a number of protein prenyltransferases from different sources. A X-ray crystal structure of one of these analogues bound to rat farnesyltransferase illustrates that they are good substrate mimics. Of particular importance, these new analogues can be enzymatically incorporated into Ras-based peptide substrates allowing the preparation of molecules with photoactive isoprenoids that may serve as valuable probes for the study of prenylation function. Photoaffinity labeling of human protein geranylgeranyltransferase with 32P-labeled forms of these analogues suggests that the C-10 locus of bound geranylgeranyl diphosphate (GGPP) is in close proximity to residues from the β-subunit of this enzyme. These results clearly demonstrate the utility of these compounds as photoaffinity labeling analogues for the study of a variety of protein prenyltransferases and other enzymes that employ FPP or GGPP as their substrates.
Enzymatic synthesis of farnesyl laurate in organic …
Substrate analogues for isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where the C3 methyl groups were replaced by chlorine, were synthesized and evaluated as substrates for avian farnesyl diphosphate synthase (FPPase). The IPP analogue (3-ClIPP) was a co-substrate when incubated with dimethylallyl diphosphate (DMAPP) or geranyl diphosphate (GPP) to give the corresponding chlorinated analogues of geranyl diphosphate (3-ClGPP) and farnesyl diphosphate (3-ClFPP), respectively. No products were detected in incubations of 3-ClIPP with 3-ClDMAPP. Incubation of IPP with 3-ClDMAPP gave 11-ClFPP as the sole product. Values of KM3-ClIPP (with DMAPP) and KM3-ClDMAPP (with IPP) were similar to those for IPP and DMAPP, however values of kcat for both analogues were substantially lower. These results are consistent with a dissociative electrophilic alkylation mechanism where the rate-limiting step changes from heterolytic cleavage of the carbon-oxygen bond in the allylic substrate to alkylation of the double bond of the homoallylic substrate.