Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF-3 rather than by phosphoranes

Abstract

Prior evidence supporting the direct observation of phosphorane intermediates in enzymatic phosphoryl transfer reactions was based on the interpretation of electron density corresponding to trigonal species bridging the donor and acceptor atoms. Close examination of the crystalline state of beta-phosphoglucomutase, the archetypal phosphorane intermediate-containing enzyme, reveals that the trigonal species is not PO-3 , but is MgF-3 (trifluoromagnesate). Although MgF-3 complexes are transition state analogues rather than phosphoryl group transfer reaction intermediates, the presence of fluorine nuclei in near-transition state conformations offers new opportunities to explore the nature of the interactions, in particular the independent measures of local electrostatic and hydrogen-bonding distributions using 19F NMR. Measurements on three beta-PGM-MgF-3 -sugar phosphate complexes show a remarkable relationship between NMR chemical shifts, primary isotope shifts, NOEs, cross hydrogen bond F...H-N scalar couplings, and the atomic positions determined from the high-resolution crystal structure of the beta-PGM-MgF--3 -G6P complex. The measurements provide independent validation of the structural and isoelectronic MgF--3 model of near-transition state conformations.

Fig. 1.

A ${}^{31}\mathrm{P}$ NMR spectrum of the crystallization components, including β-PGM, G6P, ${\mathrm{MgCl}}_2$ and ${\mathrm{NH}}_4\mathrm{F}$. The spectrum shows (expanded in inset) resonances of the protein-bound phosphate from G6P in the $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}\mathrm{G}6\mathrm{P}\mathrm{\text{-}}\mathrm{TSA}$ complex (5.00 ppm), free G6P in solution as α- and β-anomers (2.70 and 2.82 ppm, respectively) and free Pi (0.72 ppm), as well as minor amounts of several other non-protein-bound sugar phosphates. No other peaks are observed at chemical shifts resonating upfield of Pi characteristic of a protein-bound aspartyl phosphate or a pentacoordinate phosphorane species.

Fig. 2.

Structure of the $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}\mathrm{G}6\mathrm{P}\mathrm{\text{-}}\mathrm{TSA}$ complex active site. (A) The difference Fourier map and the anomalous substructure of the $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}\mathrm{G}6\mathrm{P}\mathrm{\text{-}}\mathrm{TSA}$ complex. Anomalous difference density contoured at 3σ is shown as a magenta mesh. A large peak (height $7.1\sigma$) is visible for the phosphorus atom in G6P. No corresponding phosphorus peak was observed in the active site confirming the assignment of the trigonal planar species as ${\mathrm{MgF}}_3^{-}$ and not ${\mathrm{PO}}_3^{-}$. The difference electron density (${\mathrm{F}}_o-{\mathrm{F}}_c$) from the same data is shown as a gray mesh contoured at 3σ for G6P and the ${\mathrm{MgF}}_3^{-}$ moiety before their inclusion in the model. (B) Schematic view of the $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}\mathrm{\text{sugar phosphate}}\mathrm{\text{-}}\mathrm{TSA}$ complex active site. Three sugar moieties were studied: G6P ($\mathrm{R}=\mathrm{OH}$, $\mathrm{X}=\mathrm{O}$); 6-deoxy-6-(phosphonomethyl)-D-glucopyranoside ($\mathrm{R}=\mathrm{OH}$, $\mathrm{X}={\mathrm{CH}}_2$); 2-deoxy-G6P ($\mathrm{R}=\mathrm{H}$, $\mathrm{X}=\mathrm{O}$).

Fig. 3.

The reported pentacovalent phosphorus intermediate with β-PGM (17) is a $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}\mathrm{G}6\mathrm{P}\mathrm{\text{-}}\mathrm{TSA}$ complex. (A) Difference Fourier maps calculated for the structure 1o08 contoured at $+3\sigma$ and $-3\sigma$. The difference maps show significant discrepancies from the original interpretation (17) in the derived bond lengths and assignment of atoms in the TBP moiety. Positive peaks (ca. $8\sigma$, green) are observed beyond each of the equatorial atoms of the TBP indicating that the assigned equatorial bond lengths were too short. The large negative peak ($9.6\sigma$, red) for the central coordinating atom indicates that the true atomic species is lighter than phosphorus. (B) Difference Fourier maps calculated after refinement against the deposited structure factors (www.pdb.org) with ${\mathrm{MgF}}_3^{-}$ replacing ${\mathrm{PO}}_3^{-}$ as the trigonal planar species. Unrestrained refinement of the deposited coordinates against the deposited structure factors leads to equatorial bond lengths of ca. 1.9 Å, which are consistent with our observed bond lengths. Replacing ${\mathrm{PO}}_3^{-}$ with ${\mathrm{MgF}}_3^{-}$ as the trigonal planar species in the model eliminates peaks in the difference Fourier maps above $3\sigma$.

Fig. 4.

${}^{19}\mathrm{F}$ NMR spectra of three $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}\mathrm{\text{sugar phosphate}}\mathrm{\text{-}}\mathrm{TSA}$ complexes. Spectra were recorded at 25 °C in 50 mM ${\mathrm{K}}^{+}$ Hepes buffer at pH 7.2, in 100% ${\mathrm{H}}_2\mathrm{O}$ or in 100% ${\mathrm{D}}_2\mathrm{O}$. Chemical shifts are given in ppm for each ${}^{19}\mathrm{F}$ resonance in the complex. (A) $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}\mathrm{G}6\mathrm{P}\mathrm{\text{-}}\mathrm{TSA}$ in 100% ${\mathrm{H}}_2\mathrm{O}$ buffer ($F_A=-147.0$, $F_B=-151.8$, $F_C=-159.0$). (B) $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}\mathrm{G}6\mathrm{P}\mathrm{\text{-}}\mathrm{TSA}$ in 100% ${\mathrm{D}}_2\mathrm{O}$ buffer ($F_A=-148.6$, $F_B=-153.3$, $F_C=-159.8$). (C) $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}\mathrm{\text{phosphonate}}\mathrm{\text{-}}\mathrm{TSA}$ in 100% ${\mathrm{H}}_2\mathrm{O}$ buffer ($F_A=-147.5$, $F_B=-153.5$, $F_C=-157.4$). (D) $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}\mathrm{\text{phosphonate}}\mathrm{\text{-}}\mathrm{TSA}$ in 100% ${\mathrm{D}}_2\mathrm{O}$ buffer ($F_A=-149.1$, $F_B=-154.9$, $F_C=-158.3$). (E) $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}2\mathrm{deoxyG}6\mathrm{P}\mathrm{\text{-}}\mathrm{TSA}$ in 100% ${\mathrm{H}}_2\mathrm{O}$ buffer ($F_A=-143.5$, $F_B=-149.7$, $F_C=-177.1$). (F) $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}2\mathrm{deoxyG}6\mathrm{P}\mathrm{\text{-}}\mathrm{TSA}$ in 100% ${\mathrm{D}}_2\mathrm{O}$ buffer ($F_A=-145.2$, $F_B=-151.2$, $F_C=-177.3$) and with peak $F_A$ showing evidence of residual proton occupancy at one β-PGM hydrogen bond donor site resulting from exchange protection in the ${\mathrm{D}}_2\mathrm{O}$ buffer. Free ${\mathrm{F}}^{-}$ resonates at $-119.0\mathrm{ppm}$ in 100% ${\mathrm{H}}_2\mathrm{O}$ buffer and at $-122.0\mathrm{ppm}$ in 100% ${\mathrm{D}}_2\mathrm{O}$ buffer.

Fig. 5.

Correlation of ${}^{19}\mathrm{F}$ NMR parameters and their relationships to the crystalline state. (A) Correlation plot showing the relationship between chemical shift (ppm) and isotope shift (${\delta }_{{\mathrm{H}}_2\mathrm{O}}-{\delta }_{{\mathrm{D}}_2\mathrm{O}}$, ppm) for the ${}^{19}\mathrm{F}$ resonances of the $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}\mathrm{G}6\mathrm{P}\mathrm{\text{-}}\mathrm{TSA}$ complex (circles), the $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}\mathrm{\text{phosphonate}}\mathrm{\text{-}}\mathrm{TSA}$ complex (triangles) and the $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}2\mathrm{deoxyG}6\mathrm{P}\mathrm{\text{-}}\mathrm{TSA}$ complex (squares). Linear regression analysis gives ${\mathrm{R}}^2=0.94$. (B) Correlation plot showing the relationships between $J_{\mathrm{HF}}$ (filled symbols) and $J_{\mathrm{NF}}$ (open symbols) couplings with the corresponding internuclear distances derived from structures of the $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{MgF}}_3\mathrm{\text{-}}\mathrm{G}6\mathrm{P}\mathrm{\text{-}}\mathrm{TSA}$ (circles) and $\mathrm{PGM}\mathrm{\text{-}}{\mathrm{AlF}}_4\mathrm{\text{-}}\mathrm{G}6\mathrm{P}\mathrm{\text{-}}\mathrm{TSA}$ (squares) (10) complexes. The F-N distances are derived directly from the experimental coordinates, and the F-H distances are determined to hydrogens positioned using the program XPLOR.