Many enzymes require electrophilic or oxidative post-translational modifications or exogenous cofactors for catalysis. While these moieties confer function and are highly regulated, they are not predictable from genetic sequence and represent an underexplored hemisphere of biology. To date, chemical proteomics technologies such as activity-based protein profiling (ABPP) have been successful in identifying enzymes, inhibitors and drugs for the intrinsically nucleophilic proteome. Here we expanded the recent reverse- polarity (RP)-ABPP methodology to explore electrophilic-dependent metabolic enzymes in vivo and in bacteria and present two main stories of two enzymes with similar electrophilic cofactors in two different organisms and diseases. In the first, we annotated two cofactor-dependent metabolic reductases of the energy generating Stickland fermentation in Clostridioides difficile, the pathogenic bacterium of the gut microbiome responsible for C. difficile infection (CDI). We structurally characterized the pyruvoyl cofactors of D-proline and glycine reductase and showed their activity is context-dependent and regulated by their amino acid substrates. Further, we demonstrated that D-proline reductase is consistently active across toxigenic strains of C. difficile and can be therapeutically modulated by small molecules. Ultimately, as proline utilization is vital for C. difficile during colonization, we suggest that D-proline reductase inhibition will limit C. difficile’s ability to compete with other microbiota for nutrients, allowing repopulation of the microbiome and clearance of infection. In the second, we explored the novel glyoxylyl group of Secernin-3 and performed biochemical characterization that disproves predicted hydrolase activity, shows glyoxylyl conservation across isoforms and orthologues, and confers functionality of this group for enzyme function. Additionally, in terms of functional characterization, we paired genetic tools with chemical proteomics to demonstrate that glyoxylyl-SCRN3 is a potential brain specific enzyme with a conserved genetic consequence in thermal nociception. Together these studies demonstrate the unexplored power of electrophilic functionality in the proteome and the utility of RP-ABPP to characterize metabolic activities in diseases from the gut to the brain.