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 (eIF2). Recent studies in the yeast Saccharomyces cerevisiae have served to characterize genetically the exchange factor. However, enzyme kinetic studies of the yeast enzyme have been hindered by the lack of sufficient quantities of protein suitable for biochemical analysis. We have purified yeast eIF2B 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 degrees C. The calculated turnover number (K(cat)) of 43.2 pmol of GDP released per min/pmol of eIF2B at 30 degrees C is approximately 1 order of magnitude lower than values previously reported for the mammalian factor. Reciprocal plots at varying fixed concentrations of the second substrate were linear and intersected to the left of the y axis. This is consistent with a sequential catalytic mechanism and argues against a ping-pong mechanism similar 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.
Nika
J
J
Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75080, USA.
Yang
W
W
Pavitt
G D
GD
Hinnebusch
A G
AG
Hannig
E M
EM
D015153:Blotting, Western; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D020717:Eukaryotic Initiation Factor-2B / Q000302:isolation & purification* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000737:chemistry*
D020717:Eukaryotic Initiation Factor-2B; D006153:Guanosine Diphosphate