Transition-state analysis of S. pneumoniae 5′-methylthioadenosine nucleosidase

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′-³H], [1′-¹⁴C], [2′- ³H], [4′-³H], [5′-³H₂], [9-¹⁵N] and [Me-³H₃] MTAs. The intrinsic KIEs suggest an S_N1 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′-C2′-H2′ dihedral angle of 70°. Ab initio Hartree-Fockand 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′-³H KIE arises from hyperconjugation between the lonepair (n_p) of O4′ and the σ* (C4′-H4′) antibonding orbital owing to polarization of the 3′-hydroxyl by Glu174. A [methyl-³H₃] KIE is due to hyperconjugation between n_p of sulfur and σ* of methyl C-H bonds. The van der Waal contacts increase the 1′-³H KIE because of induced dipole-dipole interactions. The 1′-³H 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 10³-fold decrease in the catalytic efficiency of S. pneumoniae MTAN relative to that from E. coli.