title: Isopentenyl-diphosphate isomerase: irreversible inhibition by 3-methyl-3,4-epoxybutyl diphosphate.
abstract: Isopentenyl-diphosphate:dimethylallyl-diphosphate isomerase (EC 5.3.3.2) catalyzes the 1,3-allylic rearrangement of the homoallylic substrate isopentenyl diphosphate (IPP) to its allylic isomer, dimethylallyl diphosphate (DMAPP). Incubation of yeast IPP isomerase with 3-methyl-3,4-epoxybutyl diphosphate (EIPP) resulted in a time-dependent first-order loss of activity characteristic of an active-site-directed irreversible process, where k2 = 0.63 +/- 0.10 min-1 and KI = 0.37 +/- 0.11 microM. A 1:1 covalent E-I complex was formed upon incubation with [1-14C]EIPP. The inhibited enzyme was treated with trypsin to give two radioactive fragments, which were purified by reversed-phase HPLC on a C18 column. The modified amino acid in each fragment was identified as C139 by sequencing the radiolabeled peptides. Incubation of IPP isomerase with [2,4,5-13C3]EIPP gave a 13C-labeled E-I complex. A 1H-13C heteronuclear multiquantum correlation spectrum had strong cross-peaks at 1.2/28 and 2.9/48 ppm, which we assigned to the labeled methyl group and C(4) methylene, respectively, of the inhibitor. In addition, a weak signal at 2.17/42 ppm may be from the C(2) methylene. Comparison of these chemical shifts with those of a synthetic adduct isolated from treatment of EIPP with cysteine indicates C139 attacks C(4) of EIPP to generate a thioether linkage between the enzyme and the inhibitor.
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City 84112.
authors.forename: X J
authors.initials: XJ
authors.lastname: Lu
authors.forename: D J
authors.initials: DJ
authors.lastname: Christensen
authors.forename: C D
authors.initials: CD
authors.lastname: Poulter
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D019748:Carbon-Carbon Double Bond Isomerases; D002851:Chromatography, High Pressure Liquid; D004852:Epoxy Compounds / Q000494:pharmacology*; D045782:Hemiterpenes; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014357:Trypsin / Q000378:metabolism
chemical_list: D004852:Epoxy Compounds; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; C077385:3-methyl-3,4-epoxybutyl diphosphate; D014357:Trypsin; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase