AbstractInEscherichia coli, inconsistencies betweenin vitrotRNA aminoacylation measurements andin vivoprotein synthesis demands were postulated almost 40 years ago, but have proven difficult to confirm. Whole-cell modeling can test whether a cell behaves in a physiologically correct manner when parameterized within vitromeasurements by providing a holistic representation of cellular processesin vivo. Here, a mechanistic model of tRNA aminoacylation, codon-based polypeptide elongation, and N-terminal methionine cleavage was incorporated into a developing whole-cell model ofE. coli. Subsequent analysis confirmed the insufficiency of aminoacyl-tRNA synthetase kinetic measurements for cellular proteome maintenance, and estimated aminoacyl-tRNA synthetasekcats that were on average 7.6-fold higher. Simulating cell growth with perturbed kcats demonstrated the global impact of thesein vitromeasurements on cellular phenotypes. For example, an insufficientkcatfor HisRS caused protein synthesis to be less robust to the natural variability in aminoacyl-tRNA synthetase expression in single cells. More surprisingly, insufficient ArgRS activity led to catastrophic impacts on arginine biosynthesis due to underexpressed N-acetylglutamate synthase, where translation depends on repeated CGG codons. Overall, the expandedE. colimodel deepens understanding of how translation operates in anin vivocontext.