Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase - PubMed (original) (raw)

. 2008 Feb 12;105(6):1844-8.

doi: 10.1073/pnas.0711659105. Epub 2008 Feb 4.

Federico Ruiz, Raul Cachau, Isabelle Hazemann, Flora Meilleur, Andre Mitschler, Stephan Ginell, Pavel Afonine, Oscar N Ventura, Alexandra Cousido-Siah, Michael Haertlein, Andrzej Joachimiak, Dean Myles, Alberto Podjarny

Affiliations

Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase

Matthew P Blakeley et al. Proc Natl Acad Sci U S A. 2008.

Abstract

We present results of combined studies of the enzyme human aldose reductase (h-AR, 36 kDa) using single-crystal x-ray data (0.66 A, 100K; 0.80 A, 15K; 1.75 A, 293K), neutron Laue data (2.2 A, 293K), and quantum mechanical modeling. These complementary techniques unveil the internal organization and mobility of the hydrogen bond network that defines the properties of the catalytic engine, explaining how this promiscuous enzyme overcomes the simultaneous requirements of efficiency and promiscuity offering a general mechanistic view for this class of enzymes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

X-ray results. (A) Ribbon drawing of hydrogenated (S1) (red) and deuterated (S2) (blue) structures (rmsd = 0.7 Å), superposed with x-ray 2_F_o−_F_c density map (S2) structure, 1.5 rms magenta contours for the inhibitor IDD594. (B) Active-site conformation showing the residues around NADP+ and inhibitor IDD594 for the deuterated structure (S2). (C) Closeup of Lys-77 and Asp-43 in deuterated h-AR superposed with the two difference maps [deuterated (S2), 2 rms blue contours; and hydrogenated (S1), 2 rms red contours]. These maps show the hydrogen atom in the H-bond Asp-43–Lys-77 in two alternative conformations. This shared partial protonation is indicated also by the bond lengths (corresponding to structure S2) in the carboxylate of Asp-43. The H-atom is placed on the Lys-77 side.

Fig. 2.

Fig. 2.

Comparison of x-ray and neutron results. (A) X-ray model of fully deuterated h-AR-IDD594 complex [0.80-Å data collected at 15K, S2; treatment with HKL2000 (16); refinement with SHELX (17)] superposed with electron density difference map (_F_o−_F_c, 2 rms blue contours, phases calculated from model without deuteriums). The map suggests partial deuteration of the Asp-43. The model shows the neutral state for the Asp-43-Lys-77 pair. (B) Model from joint x-ray/neutron refinement of fully deuterated h-AR-IDD594 complex (S3) (both data collected at room temperature) superposed with neutron scattering-density map calculated with phases from the model with all deuteriums (2_F_o−_F_c, 2 rms red contours, 1 rms gold contours, S3). Note that the deuterium atom D (marked in magenta) was included in the model but is only weakly present in the map, confirming the partial protonation of the Asp-43-Lys-77 pair. (C) Closeup of B centered on the Lys-77 head. The map (2 rms red contours) shows strong density for only the two deuteriums marked in light gray.

Fig. 3.

Fig. 3.

QM/MD results. (A–C) Three steps of the QM/MD calculations before (A), during (B), and after (C) PT. The calculations suggest that the presence of the charged nicotinamide head has a marked influence in the behavior of the reaction centre region. The initial hydride transfer step provokes a driving force for PT, because of both the charge on the substrate and the effect on the Lys component of the salt bridge, which donates back a proton to Asp to reduce its positive charge, thus triggering the movement of Tyr and the consequent PT. Note the shortening of the distance between Lys 77 and Tyr-48 between frames A and B. (D) Map corresponding to PT step (B) computed using quantum chemical methods. The image shows the important residues in the pathway, especially Asp-43, Lys-77, and Tyr-48, as well as the substrate. Note the density (red arrow) around the oxygen atom of Tyr-48 extending over the Lys-77 nitrogen, in good agreement with the experiment.

Fig. 4.

Fig. 4.

Proposed reaction pathway. (A) I–VI show the proposed reaction pathway. VII and VIII show the proton donation for a “charged” model, in which both Lys-77 and Asp-43 are charged. VI* shows the complex with the inhibitor IDD 594. (B) Energies calculated for each reaction step. The proposed pathway I–VI is shown in black (before hydride donation) and blue (after hydride donation) lines. The “charged model” pathway is included (red lines) after hydride donation (VII–VIII–VI). Note that for the proposed model, the proton donation is barrierless (blue lines), whereas for the “charged model,” there is an energy barrier (red lines).

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