Double prenylation by RabGGTase can proceed without dissociation of the mono-prenylated intermediate

J Biol Chem. 2001 Dec 28;276(52):48631-6. doi: 10.1074/jbc.M106470200. Epub 2001 Oct 8.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkyl and Aryl Transferases / metabolism*
  • Animals
  • Chromatography, High Pressure Liquid / methods
  • Dansyl Compounds / metabolism
  • Energy Transfer / physiology
  • Indicators and Reagents / metabolism
  • Kinetics
  • Multienzyme Complexes
  • Polyisoprenyl Phosphates / chemistry
  • Protein Binding
  • Protein Prenylation*
  • Spectrometry, Fluorescence / methods
  • Spectrometry, Mass, Electrospray Ionization
  • rab GTP-Binding Proteins / metabolism*

Substances

  • Dansyl Compounds
  • Indicators and Reagents
  • Multienzyme Complexes
  • Polyisoprenyl Phosphates
  • rab7 protein
  • Alkyl and Aryl Transferases
  • Rab geranylgeranyltransferase
  • rab GTP-Binding Proteins
  • geranylgeranyl pyrophosphate