| Abstract
| - Geranylgeranyltransferase type II (GGTase-II) modifies small monomeric GTPases of the Rabfamily by attaching geranylgeranyl moieties onto two cysteines of their C-terminus. We investigated towhat extent GGTase-II discriminates between its native substrate geranylgeranyl pyrophosphate (GGpp)and other phosphoisoprenoids, including farnesyl pyrophosphate (Fpp). On the basis of a novel fluorescentassay, we demonstrated that GGpp binds to GGTase-II with an affinity of 8 ± 4 nM, while Fpp is boundless strongly (Kd = 60 ± 8 nM). Analysis of the binding kinetics of four different phosphoisoprenoidsindicated that in all cases association is rapid, with rate constants in the range of 0.15 nM-1 s-1. Incontrast, the dissociation rates differed greatly, depending on the phosphoisoprenoid used, with weakbinding substrates generally displaying an increased rate of dissociation. The affinity of GGpp and Fppfor GGTase-II was also determined in the presence of the Rab7−REP-1 complex. The affinity for GGppwas essentially unaffected by the presence of the complex; Fpp on the other hand bound less strongly tothe GGTase-II under these conditions, resulting in a Kd of 260 ± 60 nM. In vitro prenylation experimentswere used to establish that Fpp not only does bind to GGTase-II but also is transferred with an observedrate constant of 0.082 s-1 which is very similar to that of GGpp. The implications of the low level ofdiscrimination by GGTase-II for the in vivo specificity of the enzyme and the use of farnesyltransferaseinhibitors in anti-cancer therapy are discussed.
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