Browsing by Subject "Caged compounds"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    Applications of substrate analogues for studies of prenyltransferase enzymes.
    (2010-04) DeGraw, Amanda Jane
    Prenyltransferase enzymes serve a variety of important biological functions from the creation of natural rubber to the modification of signal transduction proteins. The focus of this thesis is twofold; to understand better the isoprenoid chain elongation prenyltransferase enzymes, which catalyze the extension of isoprene units, and the protein prenyltransferases, which catalyze the transfer of an isoprenyl diphosphate to a protein or peptide. Substrate analogues have proven to be versatile tools for probing the identity, structure, mechanism, and function of various prenyltransferase enzymes. Described here are a variety of analogues of prenyltransferase substrates towards the study of prenyltransferase enzymes. A photoactive phosphonophosphate-containing analogue of farnesyl diphosphate (FPP) that can covalently modify proteins it is bound to upon irradiation with light was characterized and applied towards the identification of cis-prenyltransferase protein(s) involved in rubber biosynthesis. Kinetic and structural studies with this analogue and protein farnesyltransferase (PFTase) or protein geranylgeranyltransferase type-I (PGGTase-I) demonstrate that this probe is a good mimic of isoprenoid diphosphates. The phosphonophosphate linkage resulted in enhanced stability of the analogues, allowing it to be used as a label and identify a specific protein in rubber biosynthesis, rubber elongation factor (REF). The cross-linking of REF with a photoactivatible analogue of FPP suggests that REF can interact with isoprenoid diphosphates during rubber biosynthesis and this interaction may be key for the process. However, results indicated it is unlikely that REF is the sole protein responsible for rubber synthesis, thus prompting further work in the area. A caged compound is a biologically relevant molecule rendered inactive by a link to a chemical group (the "cage") through a photolabile bond. A series of photoactivatable protein prenyltransferase substrate analogues were created to achieve temporal control of prenyltransferase activity. Detailed characterization of these probes was performed to explore their applicability in protein prenylation studies. The first generation of caged PFTase analogues contain a nitrobenzyl-based photolabile group incorporated at the distal phosphate of the isoprenoid diphosphate substrate or the sulfhydryl side-chain of the cysteine residue in a CAAX peptide substrate. Kinetic studies of caged isoprenoid diphosphates demonstrated that they are poor substrates for PFTase but, upon irradiation, can efficiently release FPP upon irradiation which can be utilized for catalysis. The caged CAAX peptide photo releases the parent peptide with similar kinetics to the caged isoprenoid diphosphates. When caged the CAAX peptide does not function as a substrate, but is able to bind with efficient capacity and nearly identical conformation as compared to the photo-released peptide and does not interfere with the binding of the isoprenoid diphosphate substrate. These results lead to a wide variety of experiments where temporal control over protein prenylation is necessary. A second set of isoprenoid diphosphate analogues was created, bearing an azide or alkyne moiety. These analogues were applied as chemical proteomic probes for studying the mammalian protein prenylome. Cells were treated with either the alcohol, which is converted into the diphosphate by cellular kinases or the diphosphate isoprenoid analogue. These analogues were then appended onto prenylated proteins and through Cu(I)-catalyzed cycloaddition with a corresponding azide- or alkyne-modified fluorophore, direct visualization of prenylated proteins was accomplished. Application of this same reaction with a biotinylated capture reagent allowed for enrichment of the modified proteins and subsequent identification by liquid chromatography-tandem mass spectrometry (LC-MS). This work is still in progress.