PMID: 2491852Jan 5, 1989Paper

Mechanism of the nucleotide exchange reaction in eukaryotic polypeptide chain initiation. Characterization of the guanine nucleotide exchange factor as a GTP-binding protein.

The Journal of Biological Chemistry
J N Dholakia, A J Wahba

Abstract

Polypeptide chain initiation in mammalian systems is regulated at the level of the guanine nucleotide exchange factor (GEF). This multisubunit protein catalyzes the exchange of GDP bound to eukaryotic initiation factor 2 (eIF-2) for GTP. Although various models have been proposed for its mode of action, the exact sequence of events involved in nucleotide exchange is still uncertain. We have studied this reaction by three different experimental techniques: (a) membrane filtration assays to measure the release of [3H]GDP from the eIF-2.[3H]GDP binary complex, (b) changes in the steady-state polarization of fluorescamine-GDP during the nucleotide exchange reaction, and (c) sucrose gradient analysis of the total reaction. The results obtained do not support the reaction as written: eIF-2.GDP + GEF in equilibrium eIF-2.GEF + GDP. The addition of GEF alone does not result in the displacement of eIF-2-bound GDP. The release of bound GDP is dependent on the presence of both GTP and GEF, and this argues against the possibility of a substituted enzyme (ping-pong) mechanism for the guanine nucleotide exchange reaction. An important finding of the present study is the observation that GTP binds to GEF. The Kd value of 4 microM for GTP was ...Continue Reading

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