Phosphoisoprenoid binding specificity of geranylgeranyltransferase type II

Geranylgeranyltransferase type II (GGTase-II) modifies small monomeric GTPa ses of the Rab family by attaching geranylgeranyl moieties onto two cystein es of their C-terminus. We investigated to what extent GGTase-II. discrimin ates between its native substrate geranylgeranyl pyrophosphate (GGpp) and o ther phosphoisoprenoids, including farnesyl pyrophosphate (Fpp). On the bas is of a novel fluorescent assay, we demonstrated that GGpp binds to GGTase- II with an affinity of 8 +/- 4 nM, while Fpp is bound less strongly (K-d = 60 +/- 8 nM). Analysis of the binding kinetics of four different phosphoiso prenoids indicated that in all cases association is rapid, with rate consta nts in the range of 0.15 nM(-1) s(-1). In contrast, the dissociation rates differed greatly, depending on the phosphoisoprenoid used, with weak bindin g substrates generally displaying an increased rate of dissociation. The af finity of GGpp and Fpp for GGTase-II was also determined in the presence of the Rab7-REP-1 complex. The affinity for GGpp was essentially unaffected b y the presence of the complex; Fpp on the other hand bound less strongly to the GGTase-II under these conditions, resulting in a K-d of 260 +/- 60 nM. In vitro prenylation experiments were used to establish that Fpp not only does bind to GGTase-II but also is transferred with an observed rate consta nt of 0.082 s(-1) which is very similar to that of GGpp. The implications o f the low level of discrimination by GGTase-II for the in vivo specificity of the enzyme and the use of farnesyltransferase inhibitors in anti-cancer therapy are discussed.