title: Allosteric regulation of substrate binding and product release in geranylgeranyltransferase type II.
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 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.
authors.affiliation: Max-Plank-Institute for molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
authors.forename: N H
authors.initials: NH
authors.lastname: Thomä
authors.forename: A
authors.initials: A
authors.lastname: Iakovenko
authors.forename: A
authors.initials: A
authors.lastname: Kalinin
authors.forename: H
authors.initials: H
authors.lastname: Waldmann
authors.forename: R S
authors.initials: RS
authors.lastname: Goody
authors.forename: K
authors.initials: K
authors.lastname: Alexandrov
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000378:metabolism*; D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D005456:Fluorescent Dyes / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017368:Protein Prenylation; D051381:Rats; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D017186:Titrimetry; D062367:ortho-Aminobenzoates / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C513166:Chm protein, rat; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D011106:Polyisoprenyl Phosphates; D062367:ortho-Aminobenzoates; D000090802:rab7 GTP-Binding Proteins; C000716466:rab7 GTP-binding proteins, rat; C065197:3'-(methylanthraniloyl)-2'-deoxy-guanosine diphosphate; D006153:Guanosine Diphosphate; D019883:Alkyl and Aryl Transferases; C075607:Rab geranylgeranyltransferase; D020691:rab GTP-Binding Proteins; C002963:geranylgeranyl pyrophosphate