Thermodynamic Characterization of the Binding of Nucleotides to Glycyl-tRNA Synthetase

The interaction of adenine nucleotides with glycyl-tRNA synthetase was examined by several experimental approaches. ATP and nonsubstrate ATP analogues render glycyl-tRNA synthetase more resistant to digestion by a number of proteases (thrombin, Arg-C, and chymotrypsin) at concentrations that correlate with their Michaelis constants or inhibition constants, consistent with their exerting an effect by binding at the ATP site. Glycine had little effect alone but potentiated the effect of ATP in increasing the resistance to thrombin digestion, consistent with the formation of an enzyme-bound adenylate. No protection from thrombin digestion was afforded by tRNAgly. Binding constants were determined by isothermal titration calorimetry at 25 °C for ATP (2.5 × 105 M-1), AMPPNP (3.7 × 105 M-1), and AMPPCP (2.2 × 106 M-1). The nucleotides had similar values for ΔH (−71 kJ mol-1), with values for TΔS that accounted for the differences in the binding constants. Near-ultraviolet CD spectra of the enzyme−nucleotide complexes indicate that the nucleotides are bound in the anti configuration. A glycyl-adenylate analogue, glycine sulfamoyl adenosine (GSAd), bound with a large value for ΔH (−187 kJ mol-1), which was balanced by a large TΔS term to give a binding constant (3.7 × 106 M-1) only slightly larger than that of AMPPCP. Glycine binding to the enzyme could not be detected calorimetrically, and its presence did not change the thermodynamic parameters for binding of AMPPCP. AMPPNP and AMPPCP were not substrates for glycyl-tRNA synthetase. Analysis of the temperature dependence of ATP binding indicated that the heat capacity change is small, whereas the binding of GSAd is accompanied by a large negative heat capacity change (−2.6 kJ K-1 mol-1). Titrations performed in buffers with different ionization enthalpies indicate that the large value for ΔH for the adenylate analogue does not arise from a coupled protonation event. Differential scanning calorimetry indicated that glycyl-tRNA synthetase is stabilized by nucleotides. Unfolding of the protein is irreversible, and thermodynamic parameters for unfolding could therefore not be determined. The results are consistent with a significant conformational transition in glycyl-tRNA synthetase coupled to the binding of GSAd.