title: Isopentenyl diphosphate isomerase catalyzed reactions in D2O: product release limits the rate of this sluggish enzyme-catalyzed reaction.
abstract: The E. coli isopentenyl diphosphate isomerase (IDI) catalyzed reaction of isopentenyl diphosphate (IPP) in D(2)O gives a 66% yield of dimethylallyl diphosphate labeled with deuterium at the (E)-methyl group (d-DMAPP) and a 34% yield of IPP labeled with 1 mol of deuterium at C-2 (d-IPP). This shows that the release to D(2)O of the initial product of the IDI-catalyzed reaction (d-DMAPP) is slower than its conversion to d-IPP. Product dissociation is therefore rate determining for isomerization of IPP with a rate constant k(dis) ≈ k(cat) = 0.08 s(-1). The data provide an estimated rate constant of k(as) = 6 × 10(3) M(-1) s(-1) for binding of DMAPP to E. coli IDI that is similar to rate constants determined for the binding of N-protonated 2-amino ethyl diphosphate intermediate analogs to IDI from yeast [Reardon, J. E.; Abeles, R. H. Biochemistry1986, 25, 5609-5616]. We propose that ligand binding to IDI is relatively slow because there is a significant kinetic barrier to reorganization of the initial encounter complex between enzyme, substrate, and an essential Mg(2+) to form the Michaelis complex where the metal cation bridges the protein and the substrate diphosphate group.
authors.affiliation: Department of Chemistry, University at Buffalo, Buffalo, New York 14260, USA.
authors.forename: Venkatadurga
authors.initials: V
authors.lastname: Jonnalagadda
authors.forename: Krisztina
authors.initials: K
authors.lastname: Toth
authors.forename: John P
authors.initials: JP
authors.lastname: Richard
mesh_terms: D019748:Carbon-Carbon Double Bond Isomerases / Q000378:metabolism*; D002384:Catalysis; D017666:Deuterium Oxide; D004926:Escherichia coli / Q000201:enzymology; D045782:Hemiterpenes / Q000378:metabolism; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000378:metabolism
chemical_list: D045782:Hemiterpenes; D009943:Organophosphorus Compounds; C004809:isopentenyl pyrophosphate; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase; D017666:Deuterium Oxide