Thomä N H, Iakovenko A, Kalinin A, Waldmann H, Goody R S, Alexandrov K
Max-Plank-Institute for molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Biochemistry. 2001 Jan 9;40(1):268-74. doi: 10.1021/bi002034p.
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 with their 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 and product 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 study the effect of GGpp on interaction of the enzyme with its product, we generated semisynthetic doubly prenylated Rab7 bearing a fluorescent reporter group. Using this novel compound, we demonstrated that the affinity of doubly prenylated Rab7:REP-1 complex for GGTase-II was 2 and 18 nM in the absence and presence of GGpp, respectively. The difference in affinities originates mainly from a difference in the dissociation rates. Thus, binding of the new isoprenoid substrate molecule facilitates the product release by GGTase-II. The affinity of GGpp for the prenylated Rab7:REP-1:GGTase-II was K(d) = 22 nM, with one molecule of GGpp binding per molecule of prenylated ternary complex. We interpreted this finding as an indication that the geranylgeranyl moieties transferred to Rab protein do not occupy the GGpp binding site of the GGTase-II. In summary, these results demonstrate that GGpp acts as an allosteric activator that stabilizes the Rab7:REP-1:GGTase-II complex and triggers product release upon prenylation, preventing product inhibition of the enzyme.
Rab家族的GTP酶是真核细胞中囊泡运输的关键组成部分。香叶基香叶基部分的翻译后附着对于Rab功能至关重要。II型香叶基香叶基转移酶（GGTase-II）催化Rab蛋白与护送蛋白（REP）形成复合物后对其进行修饰。异戊二烯化完成后，REP和修饰后的Rab离开酶，从而开启新一轮催化。我们研究了Rab蛋白香叶基香叶基化过程中底物结合和产物释放的机制。发现Rab7:REP-1复合物与GGTase-II的结合受到香叶基香叶基焦磷酸（GGpp）的强烈调节。在GGpp存在的情况下，GGTase-II对Rab7:REP-1复合物的亲和力从约120 nM增加到约2 nM。为了研究GGpp对酶与其产物相互作用的影响，我们生成了带有荧光报告基团的半合成双异戊二烯化Rab7。使用这种新型化合物，我们证明在不存在和存在GGpp的情况下，双异戊二烯化Rab7:REP-1复合物对GGTase-II的亲和力分别为2 nM和18 nM。亲和力的差异主要源于解离速率的差异。因此，新的类异戊二烯底物分子的结合促进了GGTase-II的产物释放。GGpp对异戊二烯化Rab7:REP-1:GGTase-II的亲和力为K(d)=22 nM，每个异戊二烯化三元复合物分子结合一分子GGpp。我们将这一发现解释为表明转移到Rab蛋白上的香叶基香叶基部分没有占据GGTase-II的GGpp结合位点。总之，这些结果表明GGpp作为变构激活剂，稳定Rab7:REP-1:GGTase-II复合物并在异戊二烯化后触发产物释放，防止产物对酶的抑制。
