Tryptophanyl-transfer ribonucleic-acid synthetase from beef pancreas. Ligand binding and dissociation equilibrium between the active dimeric and inactive monomeric structures
Iborra, F.; Dorizzi, M.; Labouesse, J.
European Journal of Biochemistry 39(1): 275-282
1973
ISSN/ISBN: 0014-2956
PMID: 4770797
DOI: 10.1111/j.1432-1033.1973.tb03124.x
Accession: 017567171
Binding of tryptophan, tryptamine and Atp to tryptophanyl-t Rna synthetase was studied by equilibrium dialysis experiments. There are two binding sites per mole of enzyme both for tryptophan and for tryptamine. Ks for tryptophan is 0.95 μM and for tryptamine is 1.8 μM. In the case of Atp no binding could be measured over the range of concentrations examined. The Scatchard plots of tryptophan and tryptamine binding do not shown any cooperativity between the subunits. Dissociation of the dimeric enzyme was studied at very low protein concentration. Upon dilution of the enzyme both the [32P]PPi-Atp isotope exchange activity and the t Rna charging activity are lost simultaneously. Kinetic evidence demonstrates that the inactivation is primarily due to the dissociation of the active dimer into inactive monomers. The dissociation is strongly promoted by alkaline p H and is inhibited by the presence of tryptophan or tryptophanyladenylate but not by that of t RNA. The dissociation constant of the dimer-monomer equilibrium at p H 8.5 is 15 n M at 25 °C. The dissociation form is more susceptible to denaturation than the dimeric species. The results reported in the paper suggest that even if there are not binding interactions between the subunits, the active conformation of the enzyme depends on their associated state.