title: Quantitative Analysis of Guanine Nucleotide Exchange Factors (GEFs) as Enzymes.
abstract: The proteins that possess guanine nucleotide exchange factor (GEF) activity, which include about ~800 G protein coupled receptors (GPCRs),115 Arf GEFs,281 Rho GEFs,38 Ras GEFs,4and others for other families of GTPases,5catalyze the exchange of GTP for GDP on all regulatory guanine nucleotide binding proteins. Despite their importance as catalysts, relatively few exchange factors (we are aware of only eight for ras superfamily members) have been rigorously characterized kinetically.5-13In some cases, kinetic analysis has been simplistic leading to erroneous conclusions about mechanism (as discussed in a recent review14). In this paper, we compare two approaches for determining the kinetic properties of exchange factors: (i) examining individual equilibria, and; (ii) analyzing the exchange factors as enzymes. Each approach, when thoughtfully used,14,15provides important mechanistic information about the exchange factors. The analysis as enzymes is described in further detail. With the focus on the production of the biologically relevant guanine nucleotide binding protein complexed with GTP (G•GTP), we believe it is conceptually simpler to connect the kinetic properties to cellular effects. Further, the experiments are often more tractable than those used to analyze the equilibrium system and, therefore, more widely accessible to scientists interested in the function of exchange factors.
authors.affiliation: Laboratory of Cellular and Molecular Biology; National Cancer Institute; Bethesda, MD USA.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Randazzo
authors.affiliation: Laboratory of Cellular and Molecular Biology; National Cancer Institute; Bethesda, MD USA.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Jian
authors.affiliation: Laboratory of Cellular and Molecular Biology; National Cancer Institute; Bethesda, MD USA.
authors.forename: Pei-Wen
authors.initials: PW
authors.lastname: Chen
authors.affiliation: Laboratory of Cellular and Molecular Biology; National Cancer Institute; Bethesda, MD USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhai
authors.affiliation: Laboratory of Membrane Biochemistry and Biophysics; National Institute on Alcohol Abuse and Alcoholism; Rockville, MD USA.
authors.forename: Olivier
authors.initials: O
authors.lastname: Soubias
authors.affiliation: Laboratory of Membrane Biochemistry and Biophysics; National Institute on Alcohol Abuse and Alcoholism; Rockville, MD USA.
authors.forename: John K
authors.initials: JK
authors.lastname: Northup
keywords: G protein coupled receptors; GTP-binding protein; Heterotrimeric G proteins; Ras superfamily; guanine nucleotide exchange factors