rihC is one of a group of three ribonucleoside hydrolases found in Escherichia coli (E. coli). The enzyme catalyzes the hydrolysis of selected nucleosides to ribose and the corresponding base. A family of Vmax/Km kinetic isotope effects using uridine labeled with stable isotopes, such as 2H, 13C, and 15N, were determined by liquid chromatography/mass spectrometry (LC/MS). The kinetic isotope effects were 1.012+/-0.006, 1.027+/-0.005, 1.134+/-0.007, 1.122+/-0.008, and 1.002+/-0.004 for [1'-13C], [1-15N], [1'-2H], [2'-2H], and [5'-2H2] uridine, respectively. A transition state based upon a bond-energy bond-order vibrational analysis (BEBOVIB) of the observed kinetic isotope effects is proposed. The main features of this transition state are activation of the heterocyclic base by protonation of/or hydrogen bonding to O2, an extensively broken C-N glycosidic bond, formation of an oxocarbenium ion in the ribose ring, C3'-exo ribose ring conformation, and almost no bond formation to the attacking nucleophile. The proposed transition state for the prokaryotic E. coli nucleoside hydrolase is compared to that of a similar enzyme isolated from Crithidia fasciculata (C. fasciculata).