title: Isopentenyl-diphosphate isomerase: inactivation of the enzyme with active-site-directed irreversible inhibitors and transition-state analogues.
abstract: Seven analogues of isopentenyl diphosphate (1) and dimethylallyl diphosphate (2) containing fluorine, epoxy, and ammonium functional groups irreversibly inhibited isopentenyl-diphosphate:dimethylallyl-diphosphate isomerase (EC 5.3.3.2) from the mold Claviceps purpurea. Inactivation kinetics, substrate protection studies, and labeling experiments demonstrated that the analogues interacted stoichiometrically with the active site of the enzyme. Radioactive enzyme-inactivator complexes were stable to extended dialysis and treatment with chaotropic reagents. The complexes resulting from inactivation of isomerase by 3-(fluoromethyl)-3-buten-1-yl diphosphate (3) and 3,4-epoxy-3-methyl-1-butyl diphosphate (4) were also stable to ion-exchange chromatography and gel electrophoresis. Stoichiometric release of fluoride ion occurred during inactivation of isomerase with 3. This observation is consistent with SN2 or SN2' displacement of fluorine by an active-site nucleophile with concomitant covalent attachment of the inactivator to the enzyme. 2-(Dimethylamino)ethyl diphosphate (9) formed a stable noncovalent complex with isomerase with Kdis less than 1.2 x 10(-10) M. The enzyme-inhibitor complex was stable in 6 M urea, but the inhibitor was partially released upon treatment with SDS and 2-mercaptoethanol at 37 degrees C for 1 h. The results indicate that 9 is a transition-state/reactive intermediate analogue where the positively charged ammonium group mimics a tertiary carbocationic species in the enzyme-catalyzed reaction.
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City 84112.
authors.forename: M
authors.initials: M
authors.lastname: Muehlbacher
authors.forename: C D
authors.initials: CD
authors.lastname: Poulter
mesh_terms: D001665:Binding Sites / Q000187:drug effects; D019748:Carbon-Carbon Double Bond Isomerases; D055598:Chemical Phenomena; D002621:Chemistry; D002852:Chromatography, Ion Exchange; D002967:Claviceps / Q000201:enzymology*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005459:Fluorides / Q000378:metabolism; D045782:Hemiterpenes; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000378:metabolism / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000378:metabolism / Q000494:pharmacology
chemical_list: D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D000644:Quaternary Ammonium Compounds; C004809:isopentenyl pyrophosphate; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase; D005459:Fluorides