Mechanistic studies of rat protein farnesyltransferase indicate an associative transition state.
Protein farnesyltransferase is a zinc metalloenzyme that catalyzes the transfer of a 15-carbon farnesyl group to a conserved cysteine residue of a protein substrate. Both electrophilic and nucleophilic mechanisms have been proposed for this enzyme. In this work, we investigate the detailed catalytic mechanism of mammalian protein farnesyltransferase by measuring the effect of metal substitution and/or substrate alterations on the rate constant of the chemical step. Substitution of cadmium for the active site zinc enhances peptide affinity approximately 5-fold and decreases the rate constant for the formation of the thioether product approximately 6-fold, indicating changes in the metal-thiolate coordination in the catalytic transition state. In addition, the observed rate constant for product formation decreases for C3 fluoromethyl farnesyl pyrophosphate substrates, paralleling the number of fluorines at the C3 methyl position and indicating that a rate-contributing transition state has carbocation character. Magnesium ions do not affect the affinity of either the peptide or the isoprenoid substrate but specifically enhance the observed rate constant for product formation 700-fold, suggesting that magnesium coordinates and activates the diphosphate leaving group. These data suggest that FTase catalyzes protein farnesylation by an associative mechanism with an "exploded" transition state where the metal-bound peptide/protein sulfur has a partial negative charge, the C1 of FPP has a partial positive charge, and the bridge oxygen between C1 and the alpha phosphate of FPP has a partial negative charge. This proposed transition state suggests that stabilization of the developing charge on the carbocation and pyrophosphate oxygens is an important catalytic feature.
Huang
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Department of Biochemistry, Box 3711, Duke University Medical Center, Durham, North Carolina 27710, USA.
Hightower
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Fierke
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D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism; D005462:Fluorine Radioisotopes; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D010455:Peptides / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D017368:Protein Prenylation; D051381:Rats; D012717:Sesquiterpenes; D015032:Zinc / Q000378:metabolism
D005462:Fluorine Radioisotopes; D010455:Peptides; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D002104:Cadmium; D003035:Cobalt; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D008274:Magnesium; D015032:Zinc