Computational chemistry combined with experimentally determined multiple intrinsic kinetic isotope effects provide atomic maps of substrates at their enzymatic transition states. The transition state structures of bovine and human purine nucleoside phosphorylases (PNPs) are distinct, despite homologous catalytic sites. Specific transition state analogues have been synthesized as isozyme-specific enzymatic inhibitors. Immucillin-H is similar to the transition state of inosine in bovine PNP and has Kd values of 23 and 56 pM for bovine and human PNPs, respectively. DADMe-Immucillin-H was designed to mimic the transition state structure of 2'-deoxyinosine at the transition state of human PNP and has Kd values of 9 and 110 pM for human and bovine PNPs, respectively. These compounds are in clinical trials for leukemia and autoimmune diseases. A transition state analogue of Plasmodium falciparum PNP shows 112-fold specificity for parasite PNP relative to human PNP and kills cultured parasites. The transition state structure of human 5'-methylthioadenosine phosphorylase (MTAP) has led to pM transition state analogues with activity against solid tumors. Inhibitors designed to match the transition state structures of bacterial 5'-methylthioadenosine nucleosidases (MTANs) are active in blocking quorum sensing in cultured bacteria. Powerful inhibitors designed from transition state theory are demonstrating promise as new therapeutics for several diseases.