Inosine-uridine nucleoside hydrolase (IU-NH) catalyzes the hydrolysis of nucleosides into base and ribose moieties via a ribooxocarbenium ion transition state, which has been characterized using kinetic isotope effects. Protozoan parasites lack de novo purine and pyrimidine biosynthesis and depend on the purine salvage from the host. Vern Schramm and co-workers characterized p-aminophenyliminoribitol (pAPIR) to be a potent inhibitor of IU-NH from Crithidia fasciculata with K-d of 30 nM. The cyclic amine function of the iminoribitol ring can be either protonated (pAPIRH(+)) or unprotonated (pAPIR). pAPIRH+ resembles the charge and geometry of the ribooxocarbenium ion transition state and can be looked upon as a transition-state analogue inhibitor; however, it is known that the pAPIR species is initially bound to the enzyme. We have characterized the pAPIRH+ species as resident of the active site using ab initio calculations and molecular dynamics simulations. This is a novel use of molecular dynamics to investigate the protonation state of the bound ligand to the active site. Nanosecond molecular dynamics simulations reveal a short hydrogen-bonding network between pAPlRH(+)-O2'-Asp14-His241 triad, which is not seen in the crystal structure. Other features discussed are: hydrogen bonding between pAPIRH(+) and Asn-168, unusual geometry of the iminoribitol ring, and hydrophobic interactions.