The effect of F-actin on the binding and hydrolysis of guanine nucleotide by Dictyostelium elongation factor 1A.
Indirect evidence implicates actin as a cofactor in eukaryotic protein synthesis. The present study directly examines the effects of F-actin on the biochemical properties of eukaryotic elongation factor 1A (eEF1A, formerly EF1alpha), a major actin-binding protein. The basal mechanism of eEF1A alone is determined under physiological conditions with the critical finding that glycerol and guanine nucleotide are required to prevent protein aggregation and loss of enzymatic activity. The dissociation constants (Kd) for GDP and GTP are 2.5 microM and 0.6 microM, respectively, and the kcat of GTP hydrolysis is 1.0 x 10(-3) s-1. When eEF1A binds to F-actin, there is a 7-fold decrease in the affinity for guanine nucleotide and an increase of 35% in the rate of GTP hydrolysis. Based upon our results and the relevant cellular concentrations, the predominant form of cellular eEF1A is calculated to be GTP.eEF1A.F-actin. We conclude that F-actin does not significantly modulate the basal enzymatic properties of eEF1A; however, actin may still influence protein synthesis by sequestering GTP.eEF1A away from interactions with its known translational ligands, e.g. aminoacyl-tRNA and ribosomes.
Edmonds
B T
BT
Departments of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA. edmonds@lilly.com
Bell
A
A
Wyckoff
J
J
Condeelis
J
J
Leyh
T S
TS
D000199:Actins / Q000378:metabolism*; D000818:Animals; D004023:Dictyostelium / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013816:Thermodynamics
D000199:Actins; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D005990:Glycerol