Trichomonas vaginalis is a protozoan human parasite that causes trichomoniasis, one of the most common sexually transmitted diseases in the world. The toxicity of current therapies as well as drug resistance exhibited by certain T. vaginalis strains underscores the need for new treatment options. One possibility involves the purine salvage pathway, which is well known for its importance in organisms. Key enzymes in the protozoan pathway, one of which is purine nucleoside phosphorylase (PNP), exhibit important differences from the human homologs, indicating that inhibitors could be identified that affect TvPNP but not the human pathway, as a new class of chemotherapeutic agents against the T. vaginalis parasite. The native structure of TvPNP was determined in previous research in order to determine the basis for substrate binding and selectivity. However, the hypothesized interaction between the ligands and the key residue, Asp-204, at the active site of TvPNP was not observed. Thus, our goal is to further understand the active site of TvPNP, particularly with respect to Asp-204 in terms of substrate specificity. To do this, the crystallization of the D204N TvPNP mutants with adenosine and inosine were undertaken, and the interactions between the ligands and the enzyme were analyzed in light of kinetic data.