Protein prenyltransferases catalyze the transfer of a C15 (farnesyl) or C20 (geranylgeranyl) prenyl group from the corresponding prenyl pyrophosphate to a specific cysteine residue within a protein. Recently this type of modification has attracted considerable interest for two reactions. First, farnesylation of Ras protein is required for cellular transformation by mutant forms of the enzyme. Thus prenyltransferases may prove to be good targets for the design of new anticancer agents. Additionally, prenylation causes a dramatic change in protein hydrophobicity and may play a key role in determining the cellular destination and function of a number important regulatory proteins.
The specific aims of the proposal are: (1) Determine the chemical mechanisms of the reactions catalyzed by farnesyl and type I geranylgeranyl transferase enzymes. Enantiometrically pure C-1 deuterated prenyl pyrophosphates will be used to study the reactions catalyzed by farnesyl- and geranylgeranyl transferases. Retention, inversion, or racemization at this center will clarify the type of mechanism(s) that are operating. The prenyltransferases will also be studied by kinetic isotope effect experiments. The effect on reaction rate of deuterium substitution at C-1 of prenyl group will be determined. A value (secondary kinetic isotope effect) other than unity would be indicative of a dissociative SN1 mechanism. Heavy atom isotope effect experiments will also be performed to study the transition states of these reactions in greater detail. (2) Identify important residues involved in isoprenoid binding by the farnesyl and type I geranylgeranyl transferase enzymes. Residues involved in isoprenoid binding by farnesyl and geranylgeranyl transferases will be identified by photoaffinity labeling using two approaches. First, benzophenone-based probes will be used t label the enzymes l residues will be identified by determining the specific sites of crosslinking. Second, mutants prepared by site directed mutagenesis based on sequence alignments of prenyltransferases will be assayed for their ability to bind isoprenoids using photoactive substrate analogs. (3) Identify proteins that recognize prenylated proteins and peptides through interactions with the isoprenoid. The ability of four different proteins to recognize prenylated proteins and peptides via specific interactions with the isoprenoid will be determined. These interactions will be studied using peptides and proteins appended with photoactive isoprenoids that will be prepared either by chemical synthesis or chemoenzymatic methods. The proteins that will be studie include farnesyl transferase, isoprenlyated protein endoprotease, a-factor peptide receptor, and Ras-associated proteins.
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