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

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'-3H(2)], [9-15N] and [Me-3H(3)] 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 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'-3H 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-3H(3)] KIE is due to hyperconjugation between np of sulfur and sigma* 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 10(3)-fold decrease in the catalytic efficiency of S. pneumoniae MTAN relative to that from E. coli.

Figure 1

Hydrolysis of MTA by S. pneumoniae MTAN and the proposed transition state of the reaction. Features of the transition state that distinguish it from substrate are shown in blue.

Figure 2

Forward commitment to catalysis for the MTAN-MTA complex. The complex of S. pneumoniae MTAN and [8-¹⁴C]MTA was diluted with a large excess of unlabeled MTA at 3 sec. Subsequent reaction partitions bound ¹⁴C-MTA to product (forward commitment) or permits release into free, unbound MTA. Zero commitment extrapolates through the origin while full (∞) commitment would intersect at 1.0 on the ordinate. Forward commitment was calculated by plotting the amount of labeled adenine formed following addition of chase solution divided by amount of labeled MTA on the active site before dilution with chase solution and extrapolating this ratio to zero time. The line is drawn from an ordinary least square fit of the data, y errors only. The intercept value is 0.108 ± 0.006. The forward commitment factor is calculated from the y-intercept using the expression: (intercept/1-intercept) and is 0.121 ± 0.006 for S. pneumoniae MTAN.

Figure 3

Geometry at the H1′-C1′-C2′-H2′ torsion angle alters the 2′-³H equilibrium isotope effect (EIE) and C2′-H2′ σ bond occupancy for the transition state of S. pneumoniae MTAN (upper right panel). The atoms of the H1′-C1′-C2′-H2′ torsion angle are circled in the model (upper left panel). The isotope effects are calculated with respect to MTA.

Figure 4

Polarization of the 2′-hydroxyl by a hydroxy anion alters the 2′-³H isotope effect. The change in 2′-³H IE and C2′-H2′ bond length is shown in right panel. The model on the left shows the geometry used in the calculation. The isotope effects are calculated with respect to MTA.

Figure 5

Relative change in 2′-³H isotope effects (IEs) and C2′-H2′ bond length due to rotation of the H2′-C2′-O-H torsion angle. The isotope effects are calculated with respect to a H2′-C2′-O-H torsion angle of 90°.

Figure 6

Relative change in 1′-³H EIEs and total relative energy due to rotation of the O4′-C1′-N9-C8 torsion angle in MTA. The isotope effects are calculated with respect to MTA with an O4′-C1′-N9-C8 torsion angle of 80°.

Figure 7

Calculations showing factors affecting 1′-³H EIEs. Changes in 1′-³H EIEs and length of the C1′-H1′ bond are shown due to steric imposition of a hydrogen molecule on the C1′-H1′ bond at the transition state of S. pneumoniae MTAN. A similar result with oxygen and formaldehyde (see supporting material) is shown in the upper left panel. The variation in 1′-³H EIEs and occupancy of the p-orbital (sum of p_x, p_y, and p_z) of C1′ with altered H1′-C1′-C2′-H2′ torsion angle is shown in upper right panel. The 1′-³H EIEs in the upper panels are calculated with respect to MTA. Relative change in 1′-³ H EIEs by rotation of the H2′-C2′-O-H torsion angle at the transition state is shown in the lower left panel. The isotope effects are calculated with respect to a H2′-C2′-O-H torsion angle of 100°. The effect of polarization of the 2′-hydroxyl on relative 1′-³H EIEs is shown in the lower right panel. The IE are calculated with respect to a O^2’hydroxyl-O^anion distance of 4.0 Å.

Figure 8

Variation of 4′-³H EIEs due to polarization of the 3′-OH by a hydroxyl anion. The altered charge on the ring oxygen is shown (upper right panel). The model at the bottom shows the difference in the hyperconjugation pattern of the lone pair of ring oxygen (O4′) in MTA and at the transition state. The 4′-³H EIEs are calculated relative to MTA.

Figure 9

Rotation of C4′-C5′-S-C^Me torsion angle in tetrahydro-2-((methylthio)-methyl) furan. The tritium isotope effects of the three methyl C-Hs and the overall EIEs are summarized in the upper right panel. The overall tritium isotope effect (³H₃) for three methyl hydrogens was calculated by multiplying the individual ³H₁ isotope effect for methyl hydrogens. The geometry is show in the left panel using in a tube model. The furan ring is numbered as in MTA with C4′ being the carbon to which the 5′-methylthiogroup is attached and the methyl hydrogens are indicated as H_A, H_B and H_C.

Figure 10

The contacts made by MT-ImmA, a transition state analogue with the residues in the active site of S. pneumoniae MTAN.

Figure 11

Reaction coordinate showing the molecular electrostatic surface potential of 5′-methylthioadenosine (a substrate), the transition state and adenine with 5-methylthioribose (products). MEPs were calculated at HF/STO3G (Gaussian 98/cube) for the geometry optimized at the B1LYP/6-31G(d) level of theory and visualized with Molekel 4.0 at a density of 0.4 electron/ų.