| Abstract
| - GTPases of the Rab family are key components of vesicular transport in eukaryotic cells.Posttranslational attachment of geranylgeranyl moieties is essential for Rab function. Geranylgeranyltransferase type II (GGTase-II) catalyzes the modification of Rab proteins once they are in complex withtheir escort protein (REP). Upon completion of prenylation, REP and modified Rab leave the enzyme,enabling a new round of catalysis. We have studied the mechanism underlying substrate binding andproduct release in the geranylgeranylation of Rab proteins. Binding of the Rab7:REP-1 complex to GGTase-II was found to be strongly modulated by geranylgeranyl pyrophosphate (GGpp). The affinity of GGTase-II for the Rab7:REP-1 complex increases from ca. 120 nM to ca. 2 nM in the presence of GGpp. To studythe effect of GGpp on interaction of the enzyme with its product, we generated semisynthetic doublyprenylated Rab7 bearing a fluorescent reporter group. Using this novel compound, we demonstrated thatthe affinity of doubly prenylated Rab7:REP-1 complex for GGTase-II was 2 and 18 nM in the absenceand presence of GGpp, respectively. The difference in affinities originates mainly from a difference inthe dissociation rates. Thus, binding of the new isoprenoid substrate molecule facilitates the product releaseby GGTase-II. The affinity of GGpp for the prenylated Rab7:REP-1:GGTase-II was Kd = 22 nM, withone molecule of GGpp binding per molecule of prenylated ternary complex. We interpreted this findingas an indication that the geranylgeranyl moieties transferred to Rab protein do not occupy the GGppbinding site of the GGTase-II. In summary, these results demonstrate that GGpp acts as an allostericactivator that stabilizes the Rab7:REP-1:GGTase-II complex and triggers product release upon prenylation,preventing product inhibition of the enzyme.
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