Purification and kinetic analysis of eIF2B from Saccharomyces cerevisiae

Eukaryotic translation initiation factor 2B (eIF2B) is the heteropentameric guanine nucleotide exchange factor for translation initiation factor 2 (eI F2). Recent studies in the yeast Saccharomyces cerevisiae have served to ch aracterize genetically the exchange factor. However, enzyme kinetic studies of the yeast enzyme have been hindered by the lack of sufficient quantitie s of protein suitable for biochemical analysis. We have purified yeast eIF2 B and characterized its catalytic properties in vitro. Values for K-m and V -max were determined to be 12.2 nM and 250.7 fmol/min, respectively, at 0 d egrees C. The calculated turnover number (K-cat) of 43.2 pmol of GDP releas ed per min/pmol of eIF2B at 30 degrees C is approximately 1 order of magnit ude lower than values previously reported for the mammalian factor. Recipro cal plots at varying fixed concentrations of the second substrate were line ar and intersected to the left of the gamma axis. This is consistent with a sequential catalytic mechanism and argues against a ping-pong mechanism si milar to that proposed for EF-Tu/EF-Ts. In support of this model, our yeast eIF2B preparations bind guanine nucleotides, with an apparent dissociation constant for GTP in the low micromolar range.