Double prenylation by RabGGTase can proceed without dissociation of the mono-prenylated intermediate.
Rab geranylgeranyltransferase (RabGGTase) catalyzes the prenylation of Rab proteins. Despite possessing a single active site, RabGGTase is able to add geranylgeranyl moieties onto each of the two C-terminal cysteine residues of Rab. We have studied the kinetics of Rab double prenylation employing a combination of a novel high pressure liquid chromatography (HPLC)-based in vitro prenylation assay and fluorescence spectroscopy. Transfer of the first geranylgeranyl group proceeds with a k(1) = 0.16 s(-1), while the conversion from singly to double prenylated Rab is 4-fold slower (k(2) = 0.039 s(-1)). We found that following the first transfer reaction, the conjugated lipid is removed from the active site of RabGGTase but mono-prenylated Rab.REP complex remains bound to RabGGTase with a K(d) < 1 nm. In contrast to the doubly prenylated Rab7.REP dissociation of the mono-prenylated species from RabGGTase was only weakly stimulated by phosphoisoprenoid. Based on the obtained rate constants we calculated that at least 72% of mono-prenylated Rab molecules proceed to double prenylation without dissociating from RabGGTase. The obtained data provides an explanation of how RabGGTase discriminates between mono-prenylated intermediate and double prenylated reaction product. It also indicates that the phosphoisoprenoid acts both as a substrate and as a sensor governing the kinetics of protein.protein interactions in the double prenylation reaction.
Thomä
N H
NH
Department of Physical Biochemistry, Max-Plank-Institute for Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.
Niculae
A
A
Goody
R S
RS
Alexandrov
K
K
D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003619:Dansyl Compounds / Q000378:metabolism; D004735:Energy Transfer / Q000502:physiology; D007202:Indicators and Reagents / Q000378:metabolism; D007700:Kinetics; D009097:Multienzyme Complexes; D011106:Polyisoprenyl Phosphates / Q000737:chemistry; D011485:Protein Binding; D017368:Protein Prenylation; D013050:Spectrometry, Fluorescence / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000090802:rab7 GTP-Binding Proteins
D003619:Dansyl Compounds; D007202:Indicators and Reagents; D009097:Multienzyme Complexes; D011106:Polyisoprenyl Phosphates; D000090802:rab7 GTP-Binding Proteins; D019883:Alkyl and Aryl Transferases; C075607:Rab geranylgeranyltransferase; D020691:rab GTP-Binding Proteins; C002963:geranylgeranyl pyrophosphate