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.

Figure 1

Partial ¹H-NMR spectra of the products of IDI-catalyzed isomerization of IPP in D₂O, with homonuclear decoupling of the signals for the 1-CH₂- and 2-CH₂- of IPP by irradiation at the resonance frequency for 1-CH₂- hydrogen. The triplet at 4.35 ppm and the apparent singlets at 2.295 and 2.272 are the signals for the C-1 hydrogen of DMAPP and the C-2 hydrogen of IPP and 1, respectively. The experimental details for these ¹H NMR analyses are given in the Supporting Information.

Figure 2

Time courses for the IDI-catalyzed reactions of IPP (■) to form DMAPP_T (▼) and 1 (●). DMAPP formed at early reaction times contains one atom of –D (d-DMAPP, Scheme 3), but we observe the incorporation of multiple atoms of –D as DMAPP and IPP achieve chemical equilibrium. (A) The isomerization of 26.5 mM IPP catalyzed by 28µM E. coli IDI at pD 7.4 (20 mM imidazole), 400 mM NaCl, 10 mM MgCl₂ and 0.5 mM DTT. (B) The isomerization of 9 mM IPP catalyzed by 71 µM E. coli IDI at pD 7.4 (20 mM imidazole), 400 mM NaCl, 10 mM MgCl₂ and 0.5 mM DTT. The procedures for these experiments are given in the Supporting Information.

Scheme 1

Scheme 2

Scheme 3

Scheme 4