Abstract
Guanine nucleotide exchange factor (GEF) is a multisubunit protein involved in the initiation of translation. Although numerous models have been proposed for its mechanism of action, none have been definitive. An assay dependent on GEF activity was developed using highly purified eukaryotic initiation factor 2 (eIF-2) and GEF from Ehrlich cells. GEF was considered in terms of an enzyme whose catalytic function was the exchange of eIF-2-bound [alpha-32P]GDP for unlabeled nucleotide. The turnover number of GEF at 37 degrees C, calculated on the basis of enzyme kinetic methods is 0.027 s, which is consistent with in vivo rates of protein synthesis. Moreover, kinetic data support an enzyme-substituted mechanism as the mode of GEF function. This mechanism proposes the existence of a GEF.eIF-2.GDP complex and excludes the possibility of two guanine nucleotide binding sites on eIF-2. An analogous mechanism has been recently reported for elongation factor Ts, suggesting the importance of this mechanism to protein synthesis. The mechanism of inhibition of GEF function by eIF-2 alpha phosphorylation has also been investigated. It has been generally assumed that the mechanism by which eIF-2(P) traps GEF is an excessively stable complex, from which GEF is released very slowly. Data presented here, however, reveal that eIF-2(P).GDP is a competitive inhibitor of GEF (rather than an irreversible inhibitor) competing with eIF-2.GDP for binding to GEF. Even though the eIF-2(P).GDP.GEF complex dissociates too rapidly to measure, GEF is trapped because it has at least 150-fold greater affinity for eIF-2(P).GDP than for eIF-2.GDP. The implications of competitive inhibition with respect to the mechanism of reversal of inhibition by an eIF-2(P) phosphatase are discussed.
Highlights
An initiation factor with such guanine nucleotide exchange activity has been isolated from reticulocytes, Ehrlich cells, and HeLa cells [6,7,8,9,10,11]
The assay conditions employed were such that theconcentration of substratewas in large excess overthe enzyme concentration and, eIF-2 added with the Guanine nucleotide exchange factor (GEF) did not significantly alter theconcentration of substrate
GDP (17.5 nM) ( C and D )with (0)or without (m) 40 p~ GEF under conditions described under "Experimental Procedures." Data are expressed in the form cpmJSA, where cpm, is the measured counts/min remaining in the substrate and SA, is the specific activity of the initialsubstrate
Summary
Which strongly suggest that GEF functions via an enzymesubstituted mechanism. An analogous mechanism of action has been recently reported for elongation factor Ts [20],which suggests the importance of this mechanism to protein synthesis. The dialyzed ribosomal salt washwas clarified by centrifugation at 10,000 X g for 10 min and applied to a CM-Sephadex column equilibrated in Bo.l.The column was washed with C,, (20 mM MOPS (pH 7.6), 0.1 mM EDTA, 0.25 mM dithiothreitol, and KC1 concentration as indicated by the subscript) and eluted with cO..The peak eluted at Co. wasconcentrated uersus 1 liter of BO., plus 20% sucrose (w/v) for 17 h. The C,, peak from the CMSephadex column was, as described previously, dialyzed uersus Bo., plus 20% sucrose (w/v) for 17 h; prior to application of this fraction onto theDEAE-cellulosecolumn, the fraction was incubated with HRI, 2.5 mM ATP, and 5 mM MgAc, for 40 min at 30 'C and brought to a final concentration of mM KF. Mechanism of Guanine Nucleotide ExchangFeactor phosphorylated eIF-2present and phosphorylated eIF-2 with less than 1% unphosphorylated eIF-2 present
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