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

Vipender Singh, Vern L. Schramm

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

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 SN1 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 (np) 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 np 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.

Original languageEnglish (US)
Pages (from-to)2783-2795
Number of pages13
JournalJournal of the American Chemical Society
Volume129
Issue number10
DOIs
StatePublished - Mar 14 2007

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Isotopes
Pneumonia
adenosylhomocysteine nucleosidase
Kinetics
Hydroxyl Radical
Adenine
Polarization
Escherichia coli
Pemetrexed
5'-methylthioadenosine phosphorylase
5'-methylthioadenosine
Ions
Nucleophiles
Dihedral angle
Sulfur
Discrete Fourier transforms
Torsional stress
Conformations
Permittivity
Atoms

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Transition-state analysis of S. pneumoniae 5′-methylthioadenosine nucleosidase. / Singh, Vipender; Schramm, Vern L.

In: Journal of the American Chemical Society, Vol. 129, No. 10, 14.03.2007, p. 2783-2795.

Research output: Contribution to journalArticle

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