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Measurement of kinetic isotope effects to probe the reaction mechanism catalyzed by mammalian protein farnesyltransferase
Author(s) -
Pais June E.,
Fierke Carol A.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a275
Subject(s) - farnesyltransferase , mechanism (biology) , kinetic isotope effect , chemistry , catalysis , kinetic energy , isotope , reaction mechanism , biochemistry , enzyme , physics , prenylation , deuterium , quantum mechanics
Protein farnesyltransferase (FTase) catalyzes the transfer of a 15‐carbon prenyl group from farnesyl diphosphate (FPP) to the cysteine residue of target proteins and is a member of the newest class of zinc metalloenzymes that catalyze sulfur alkylation. Common substrates of FTase include oncogenic Ras proteins, and therefore inhibitors are under development for the treatment of various cancers. An increased understanding of the salient features of the transition state of FTase may aid in the design of potent inhibitors and enhance our understanding of the mechanism of this class of zinc enzymes. The α‐secondary kinetic isotope effect using a peptide substrate that is farnesylated rapidly is near unity, indicating that a step other than farnesylation, such as the conformational rearrangement of bound FPP, is the rate‐limiting step. For a peptide that is farnesylated slowly, this kinetic isotope effect increases to 1.154 ± 0.006, suggesting that the thiol alkylation step catalyzed by FTase proceeds via a concerted mechanism with dissociative character. The dependence of the isotope effect on the metal ion and peptide structure provide further information about the structure of the chemical transition state, as well as the conformational rearrangement of FPP. This work was supported by a National Institutes of Health Grant (GM40602) and a National Science Foundation Graduate Research Fellowship.