Yeast cells exhibit a temperature-sensitive phenotype at 37°C due to an ATP(CTP): tRNA  nucleotidyltransferase variant containing a glutamate to phenylalanine substitution at position 189.  This amino acid alteration in conserved motif C results in weakened thermal stability and reduced enzyme activity in the tRNA nucleotidyltransferase.  When the arginine at position 64 of this enzyme is changed to tryptophan, enzyme activity is increased and the temperature-sensitive phenotype is suppressed such that the yeast can grow at the restrictive temperature.  This suppressor mutation is found associated with the highly conserved motif A which plays a crucial role in catalysis.  Here the roles of glutamate 189 and arginine 64 in tRNA nucleotidyltransferase activity are explored.  The data presented here show that the decrease in enzyme activity in the enzyme bearing the glutamate to phenylalanine substitution at position 189 results from an approximately 70-fold decrease in catalytic activity (kcat) and a less than 2-fold change in apparent tRNA binding (KM).  Moreover, they show that the subsequent arginine to tryptophan substitution at position 64 results in an approximate 6-fold increase in enzyme activity as compared to the glutamate to phenylalanine substitution variant with no apparent change in KM.  Interestingly, the arginine to tryptophan substitution alone shows no major change to either apparent kcat or KM as compared to the native enzyme.  Based on these observations, molecular modeling suggests that the glutamate to phenylalanine substitution in motif C affects catalysis by altering the organization of catalytic residues in the head and neck regions of the protein.  Substitution of glutamate at position 189 by a larger amino acid produces steric repulsion that disturbs the precise positioning of motif A and B residues leading to a reduced kcat.  These data also suggest that the organization of the head and neck regions of the protein do not play a major role in tRNA binding.  Finally, the suppressor mutation resulting in the change of arginine 64 to tryptophan allows the catalytic residues of motifs A and B to be shielded from steric hindrance leading to the measured increase in kcat that restores viability at the restrictive temperature.