Significance Understanding roles and mechanisms of protein posttranslational modifications (PTMs) would greatly impact multiple scientific domains, from bioengineering to biomedical science. PTMs are known to interfere with drug action and influence biochemical networks of engineered organisms. Many PTM sites have been identified, but it remains unclear under which conditions these sites are modified. Furthermore, there is a need to understand how the cell utilizes PTMs to increase fitness. Here, we approach this challenge by integrating tools from molecular biology, biochemistry, and systems biology to unravel mechanisms through which PTMs regulate enzymes throughout Escherichia coli metabolism and demonstrate how these individual PTMs further regulate pathways and ultimately cell phenotypes. This workflow could be applied to study PTMs and their roles across species.