Kinetic isotope effects (KIEs) and computer modeling are used to approximate the transition state of S. pneumoniae 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN). Experimental KIEs were measured and corrected for a small forward commitment factor. Intrinsic KIEs were obtained for [1'-H-3], [1'-C-14], [2'-H-3], [4'-H-3], [5'-H-3(2)], [9-N-15] and [Me-H-3(3)] MTAs. The intrinsic KIEs suggest an S(N)1 transition state with no covalent participation of the adenine or the water nucleophile. The transition state was modeled as a stable ribooxacarbenium ion intermediate and was constrained to fit the intrinsic KIEs. The isotope effects predicted a 3-endo conformation for the ribosyl oxacarbenium-ion corresponding to H1'-C1'-C'-H2' dihedral angle of 70 degrees. Ab initio Hartree-Fock and DFT calculations were performed to study the effect of polarization of ribosyl hydroxyls, torsional angles, and the effect of base orientation on isotope effects. Calculations suggest that the 4'-H-3 KIE arises from hyperconjugation between the lonepair (n(p)) of O4' and the sigma* (C4'-H4') antibonding orbital owing to polarization of the 3'-hydroxyl by Glu174. A [methyl-H-3(3)] KIE is due to hyperconjugation between n(p) of sulfur and sigma* of methyl C-H bonds. The van der Waal contacts increase the 1'-H-3 KIE because of induced dipole-dipole interactions. The 1'-H-3 KIE is also influenced by the torsion angles of adjacent atoms and by polarization of the 2'-hydroxyl. Changing the virtual solvent ( dielectric constant) does not influence the isotope effects. Unlike most N-ribosyltransferases, N7 of the leaving group adenine is not protonated at the transition state of S. pneumoniae MTAN. This feature differentiates the S. pneumoniae and E. coli transition states and explains the 10(3)-fold decrease in the catalytic efficiency of S. pneumoniae MTAN relative to that from E. coli.