The cytoplasmic phenylalanine transfer ribonucleic acid ligase of Neurospora crassa is unique in that it can aminoacylate phenylalanine to the valine and alanine transfer ribonucleic acids of Escherichia coli. However, aminoacylation of these heterologous transfer ribonucleic acids is incomplete. Purified enzyme and purified transfer ribonucleic acids were used in the study of these heterologous reactions. The amount of phenylalanyl transfer ribonucleic acidVal or Ala (E. coli) formed was found to be a linear function of enzyme concentration; this was not due to irreversible enzyme inactivation. This relationship was not exhibited with homologous transfer ribonucleic acid (N. crassa) even at low levels of enzyme. Inorganic pyrophosphate at 10-8-10-7 m was found to be a potent inhibitor of both the rate and extent of phenylalanyl transfer ribonucleic acidVal or Ala (E. coli) formation in the heterologous reactions. At low synthetase concentrations the presence of inorganic pyrophosphatase resulted in a doubling of the rate and extent of phenylalanyl transfer ribonucleic acidValor AIa (E. coli) formation. Inorganic pyrophosphate at low concentrations did not have an effect on the extent of aminoacylation of transfer ribonucleic acid (N. crassa), and inorganic pyrophosphatase stimulated only slightly the rate of aminoacyl transfer ribonucleic acid production. The linear response to enzyme concentration and the incomplete aminoacylation observed in the heterologous reactions occurred in Tris-Cl buffer. In agreement with our previous findings, complete aminoacylation of transfer ribonucleic acidVal (E. coli) was observed in potassium cacodylate buffered systems. In potassium cacodylate buffer the linearity with enzyme was not seen at comparable enzyme concentrations; inorganic pyrophosphate at 10-8-10-7 m inhibited the rate but not the extent of phenylalanyl transfer ribonucleic acidVal (E. coli) production, and inorganic pyrophosphatase stimulated the rate of phenylalanyl transfer ribonucleic acidVal (E. coli) formation. The stimulation of the rate of the reaction by inorganic pyrophosphatase indicates inhibition by endogenously formed inorganic pyrophosphate. In Tris-Cl buffer, inorganic pyrophosphate acts as a competitive inhibitor with respect to both of the heterologous transfer ribonucleic acids. The Ki values for the pyrophosphate are slightly smaller than the Km's for the transfer ribonucleic acids. The presence of inorganic pyrophosphatase lowers both Km's and Ki's and indicates that the inhibition by endogenous inorganic pyrophosphate is also competitive. In contrast to the results obtained in Tris-Cl buffer, inorganic pyrophosphate acts as a noncompetitive inhibitor with respect to transfer ribonucleic acidVal (E. coli) in potassium cacodylate buffer. Apparent and VmaK values in the two buffers are comparable, but the Km to transfer ribonucleic acidVal (E. coli) in potassium cacodylate is 100-fold smaller than that in Tris-Cl. These data show that some aspects of this heterologous aminoacylation reaction observed in Tris-Cl buffer are different in potassium cacodylate buffer. An important role for inorganic pyrophosphate was demonstrated in these heterologous reactions. This inhibitor should also be considered in studying other heterologous systems, especially those carried out in Tris-Cl buffer. © 1969, American Chemical Society. All rights reserved.
