A Trojan horse transition state analogue generated by MgF3- formation in an enzyme active site

Abstract

Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. beta-Phosphoglucomutase catalyses the isomerization of beta-glucose-1-phosphate to beta-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using (19)F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF(3)(-) mimics the transferring PO(3)(-) moiety. Here we present a detailed characterization of the metal ion-fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of beta-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography.

Fig. 1.

The reaction mechanism of β-PGM and the potential enzyme stabilized species. (a) β-PGM catalyses the interconversion of β-glucose-1-phosphate (β-G1P) and β-glucose-6-phosphate (G6P) via β-glucose-1,6-bisphosphate (β-G16BP) and the phosphorylated form of the enzyme (β-PGM*). (b) The TS for the latter part of the β-PGM catalyzed reaction (i.e., β-G16BP to G6P), the proposed phosphorane INT and the TSA (MgF₃–TSA) formed from β-G6P, magnesium, and fluoride.

Fig. 2.

¹⁹F NMR spectra of the MgF₃⁻/β-PGM system. (a) The peaks labeled F_A-C (−147, −152, and −159 ppm) correspond to MgF₃⁻ bound to β-PGM in the presence of G6P. [NH₄F] = 10 mM, [MgCl₂] = 5 mM, [β-PGM] = 1 mM, [G6P] = 5 mM. Conditions: pH 7.2, [K⁺ Hepes] = 50 mM, 5°C. (b) Control with G6P omitted. (c) Control with G6P and β-PGM omitted. The peak at −119 ppm is free fluoride in solution (F⁻), and the peak at −156 ppm is MgF⁺. The three ¹⁹F resonances (−134, −135, and −154 ppm), with ≈10% of the intensity of the major peaks, most likely correspond to a minor conformer of the MgF₃–TSA complex that exchanges with the major conformer more rapidly than the complex dissociates, because these resonances correlate via saturation transfer with specific resonances of the major conformer (resonances F_A, F_B, and F_C, respectively).

Fig. 3.

Histogram of backbone amide proton chemical shift changes (Δδ) plotted against residue between β-PGM in the open form and β-PGM in MgF₃–TSA. Positive Δδ represent up-field changes for the open to closed transition. Large Δδ are expected in regions of β-PGM involved in binding substrate in the active site. However, no data were obtained for residues in the active site loops (D8–T16, L44–L53, S114–N118, V141–A142, and S171–Q172) in the open conformation, because the corresponding ¹⁵N-TROSY peaks were broadened beyond the limits of detection. This line-broadening behavior is indicative of a conformational dynamics process between two (or more) similarly populated forms, and the difference in ¹H chemical shift of ≈2 ppm between these interconverting conformations equates to conformational dynamics occurring in the millisecond timescale (i.e., dynamics in the intermediate exchange regime for ¹H). These residues are depicted with open bars. Further significant Δδ involve residues A17–Q43 positioned in two α-helices of the “cap” domain and N77–S88, which locate to the C-terminal portion of the S65–I84 α-helix and the “hinge” region (Q85–Y93). For the remainder of β-PGM, small Δδ indicate that, on formation of MgF₃–TSA, there is little change in the local protein fold outside of these regions.

Fig. 4.

The structure of the active site in MgF₃–TSA. Positions of fluoride bound to the enzyme were docked according to the 11 NOEs to the backbone amide protons. The protein structure (PDB ID code 1O08) was used as a template, and the fluorides were assigned zero van der Waals radii during their movement so that they could locate the optimum positions in the structure based solely on the NOE restraints. (a) The pale blue spheres show the results of 50 separate minimizations. (b) The active site of MgF₃–TSA. In a and b, the magnesium ion essential for catalysis is on the left.

Fig. 5.

Fluoride inhibition of catalysis by β-PGM. The reaction is inhibited by fluoride (0–10 mM) unless a high concentration of β-G16BP is present. Relative initial rates (%) are shown for the conversion of β-G1P to G6P. Reactions contained: green circles, [β-G1P] = 250 μM, [β-PGM] = 200 nM; black triangles, [β-G1P] = 250 μM, [α-G16BP] = 50 μM, [β-PGM] = 5 nM; red squares, [β-G1P] = 250 μM, [β-G16BP] = 0.5 μM, [β-PGM] = 5 nM; blue inverted triangles, ([β-G1P] = 50 μM, [β-G16BP] = 200 μM, [β-PGM] = 5 nM.