The E. coli isopentenyl diphosphate isomerase (IDI) catalyzed reaction of isopentenyl diphosphate (IPP) in D2O 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 D2O 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) approximate to k(cat) = 0.08 s(-1). The data provide an estimated rate constant of k(as) = 6 x 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. Biochemistry 1986, 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 Mg2+ to form the Michaelis complex where the metal cation bridges the protein and the substrate diphosphate group.