Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase^†

The X-ray crystal structures of human purine nucleoside phosphorylase (PNP) with bound inosine or transition-state analogues show His257 within hydrogen bonding distance of the 5‘-hydroxyl. The mutants His257Phe, His257Gly, and His257Asp exhibited greatly decreased affinity for Immucillin-H (ImmH), binding this mimic of an early transition state as much as 370-fold (Km/Ki) less tightly than native PNP. In contrast, these mutants bound DADMe-ImmH, a mimic of a late transition state, nearly as well as the native enzyme. These results indicate that His257 serves an important role in the early stages of transition-state formation. Whereas mutation of His257 resulted in little variation in the PNP·DADMe-ImmH·SO4 structures, His257Phe·ImmH·PO4 showed distortion at the 5‘-hydroxyl, indicating the importance of H-bonding in positioning this group during progression to the transition state. Binding isotope effect (BIE) and kinetic isotope effect (KIE) studies of the remote 5‘-3H for the arsenolysis of inosine with native PNP revealed a BIE of 1.5% and an unexpectedly large intrinsic KIE of 4.6%. This result is interpreted as a moderate electronic distortion toward the transition state in the Michaelis complex with continued development of a similar distortion at the transition state. The mutants His257Phe, His257Gly, and His257Asp altered the 5‘-3H intrinsic KIE to −3, −14, and 7%, respectively, while the BIEs contributed 2, 2, and −2%, respectively. These surprising results establish that forces in the Michaelis complex, reported by the BIEs, can be reversed or enhanced at the transition state

Binding of GSH at the active site of mLTC4S.

<p>A. Electron density 2fo-fc map contoured at 1.0 σ around GSH with Arg104 coordinating the sulfur in GSH. B. GSH bound at the active site, coordinated by several amino acids where the Arg51 - Tyr50 (indicated with a line) interaction in the human enzyme, is lost in the mLTC4S, which has a Phe in position 50.</p

Data collection, refinement and model building statistics of mLTC4S in complex with sulfate (apo-form), GSH and S-hexyl GSH.

a<p><i>R</i>_merge = ∑_hkl∑_i|I_i(<i>hkl</i>) – hkl)>|/∑_hkl∑_i|I_i(<i>hkl</i>)|, where I_i(<i>hkl</i>) is the intensity of the <i>i</i>th measurement of reflection <i>hkl</i> and hkl)> is the average intensity of this reflection.</p>b<p><i>R</i> = ∑ ||F_obs|–|F_calc||/∑ |F_obs|.</p>c<p><i>R_free</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096763#pone.0096763-Brunger1" target="_blank">[38]</a> was monitored with 5% of the reflection data excluded from refinement.</p>d<p>as determined by <i>MolProbity.</i></p><p>Values for the highest resolution shell are given in parentheses.</p

Steady state kinetic parameters of mLTC4S and hLTC4S against GSH and LTA₄.

<p>The enzyme activity was measured in 25mM Tris (pH 7.8), 0.1M NaCl, 0.05% DDM in the presence of either 30 µM LTA₄ and/or 5 mM GSH with 0.1 µg of enzyme.</p><p>**<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096763#pone.0096763-RinaldoMatthis2" target="_blank">[29]</a>.</p

Comparison of human and mouse LTC4S enzymes.

<p>A. Amino acid sequence alignment of human and mouse LTC4S generated with the program ClustalW. Species differences are highlighted in white. B. Mapping the amino acid differences (in red) between mouse and human trimeric LTC4S structures. The active site in one monomer is depicted with a bound GSH (green). In blue is the Phe50Tyr exchange positioned close to the active site.</p

The LTC₄ synthase reaction.

<p>A. Schematic drawing of the catalytic reaction of LTC4S where the allylic epoxide LTA₄ is conjugated with GSH at C6, to form LTC₄. B. Structure of the product analog S-hexyl GSH.</p

TK04 is a nanomolar competitive inhibitor of LTC4S.

<p>A. Chemical structure of TK04, the inhibitor used in this study. Dose-response curves for inhibition of mouse and human LTC4S by TK04. 100% activity corresponds to the enzyme activity without inhibitor, which was 44.0 µmol min⁻¹ mg⁻¹ for the mouse enzyme (red line) and 69.7 µmol min⁻¹ mg⁻¹ for the human enzyme (black line). The concentrations of substrates GSH and LTA₄ used in the assay were 5 mM and 20 µM, respectively. The IC₅₀ for mLTC4S was 135±30 nM and for the hLTC4S it was 134±16 nM.</p

Additional file 1 of Discovery of a small molecule that inhibits Bcl-3-mediated cyclin D1 expression in melanoma cells

Additional file 1: Supplementary Figure 1

Positional shift of Arg51 and loss of salt bridge at the active site of mLTC4S.

<p>A. Close up of the mLTC4S complex with SO₄²⁻, showing a shift in the position of Arg51 due to Phe50Tyr exchange. Human LTC4S is colored in green and mLTC4S is colored in gray. GSH is shown as green “lines”. *indicates that it is positioned on the neighboring subunit. B. Trimer of mLTC4S showing the amino acid exchange at position 50 where Phe in mLTC4S fails to make a salt bridge with Arg51. In hLTC4S, the Tyr50-Arg51 couple will likely contribute to trimer stability.</p

Binding of S-hexyl GSH to the active site of mLTC4S.

<p>A. The trimeric form of mLTC4S with three bound S-hexyl GSH. B. Electron density 2fo-fc map, contoured at 1.0 σ around S-hexyl GSH. C. The hydrophobic cavity with S-hexyl GSH bound (yellow stick carbons) in the hydrophobic cleft. Amino acids facing the cavity are from monomers A (yellow) and B (green).</p