The phylogenetic divergence between parasitic and mammalian phosphoglycerate mutases (PGMs) offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. With this aim we previously described ipglycermides, discovered by affinity selection from a vast nucleic acid-encoded cyclic peptide library, as the first potent and selective class of cofactor-independent PGM (iPGM) inhibitors. From C. elegans iPGM•ipglycermide co-crystal structures we now delineate the previously hypothesized metal ion binding geometries at the ipglycermide C-terminal cysteine. To develop a baseline structure-activity relationship complementing the structural details, the 14 amino acids of ipglycermide were individually substituted creating 280 DNA-encoded analogs. Binding affinities to immobilized C. elegans iPGM, measured as fold-enrichment by deep sequencing, illuminated the significance of each amino acid sidechain to the pharmacophore and guided ipglycermides designed with improved orthologous iPGM affinity. In consideration with binding kinetics, we describe how the high affinity of ipglycermide to a range of iPGM orthologs is achieved by a co-dependence on tunable metal ion coordination-associated off-rates and induced-concavity around the macrocyclic core at the phosphotransferase-phosphatase domain interface to freeze the structurally dynamic enzyme into an inactive, stable complex.