Two substrate binding sites on tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli

Tryptophanyl-tRNA synthetase of Escherichia coli has 1.8 binding sites for L-tryptophan with Kdiss of 12 x 10(-5) M as shown by equilibrium dialysis. The results are in accord with the known structure of the enzyme, and alpha2 dimer of 74,000 molecular weight, and with 2 binding sites for tryptophanyl-ATP ester. Ordinary sucrose density gradient centrifugation reveals a complex composed of one tRNATrp bound per enzyme dimer. When tRNATrp is mixed throughout the gradient at concentrations from 5.4 x 10(-6) M to 2.0 x 10(-5) M, a new peak appears in the position expected for a complex with two tRNATrp molecules bound per enzyme dimer. Sedimentation through gradients lacking tRNATrp favors dissociation of the 1:2 complex but not the 1:1 complex. The data indicate 2 binding sites for tRNATrp on tryptophanyl-tRNA synthetase.