Binding of R5P to hTK

The x-ray structure of hTK in non-covalent complex with R5P was determined to 1.2 Å resolution, refined to reasonable R values (Rwork = 12.28, Rfree = 14.59) and finally also the occupancy of R5P could be calculated (76 %). The additional positive difference electron density map found in the active site was interpreted and modeled as α-furanose form of R5P in C2-endo conformation (Fig. 64). In this conformation four atoms (ring oxygen, C1, C3 and C4) forming a plane and C2 is out of plane at the same side as C5 (tortion angle O4-C1-C3-C4 ≈ 168°). The phosphate moiety of R5P is coordinated by Arg474, Arg318, His416 and Ser345 (Fig. 65). The anomeric hydroxyl group (O1) is positioned by strong hydrogen bonding interactions with His37 and His358 and O3 is involved in a hydrogen bonding interaction with Asp424. Notably, the interaction between O2 and Gln189 is questionable in terms of hydrogen bonding geometry (not illustrated). While the phosphate moiety of R5P has a similar atomic B-factor relative to the surrounding active site residues increased B-factors are detectable for the atoms of the furanose ring which is suggestive for a higher flexibility of this part. In line with the elevated dynamics of the furanose ring is the observation that the electron density map for C5, which connects phosphate moiety and furanose ring, is diffuse. A similar observation can be made for E4P in the previous chapter suggesting similar principles for acceptor substrates binding in TK.

Fig. 64: Binding of acceptor R5P to hTK-1. X-ray structure of R5P in non-covalent complex with hTK determined to a resolution of 1.2 Å. a.) R5P bound in close proximity to the cofactor ThDP both surrounded by electron density (2mFo-DFc map, contoured at 1σ). b.) Atoms of R5P and ThDP are color-coded according to their individual B-factors with less mobile atoms in blue (8–11 Ų) and most mobile ones in red (25-28 Ų). c.) Detailed view on bound R5P. The substrate is bound as α anomer in C2 endo conformation (C2 and C5 on the same side). The side view (right) of the ligand illustrates the deviation (11.7°) of the coplanar arrangement of C1, O4, C3 and C4.

C1 C2

C3 C4

C5

O1 O4

C2 C5

11.7 ° deviation from co-planarity ThDP

a.) b.)

R5P

C2 endo rotation90 °

c.)

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Even at very low contour levels (0.5-1 σ) in a 2mFo-DFc map no fraction of β-form which contributes 63.9 % of R5P in solution (Pierce et al., 1985) is detectable. However, additional positive difference electron density peaks (mFo-DFc and 2mFo-DFc map) are observable which could be interpreted as acyclic aldehyde form of R5P. Notably, the fraction of the modeled acyclic R5P is obviously smaller than the 50 % found for the analogous EcTK-R5P complex suggesting differences for R5P binding between both TKs (Asztalos et al., 2007). Because the additional positive density peaks are diffuse modeling of the acyclic form was performed by real space refinement in COOT (Emsley et al., 2010) using a mFo-DFc residue omitted map. In this modeled but not refined position we determine a very short distance between R5Ps´ aldehyde carbon and cofactor C2 of 207 pm implicating strong repulsion in case both atoms are not covalently linked. This exceptionally long C–C bond can´t be easily explained and we have yet no clear assignment of this trapped state.

Fig. 65: Binding of acceptor R5P to hTK-2. a.) R5P is shown bound in close proximity to the cofactor ThDP (both yellow). Interactions of the acceptor substrate with active site residues are shown as blue dashed lines in addition to inter-atomic distances (in pm). b.) Acyclic R5P (cyan) that was modeled into difference electron density (mFo-DFc map contoured at 3σ) is shown as well as ThDP and cyclic R5P. Inter-atomic distances that are discussed in the text are highlighted by dashed blue lines. c.) Superposition of acyclic R5P (cyan) with the DHEThDP intermediate (purple) and the S7P-ThDP intermediate. Positional changes of R5P upon formation of S7P-ThDP are indicated (green arrows). Selected atoms are labeled.

Thus, the modeled orientation of acyclic R5P should be considered as an initial proposal. A higher resolved structure of the hTK-R5P complex is needed to clearly identify the atomic position of the minor populated acyclic aldehyde form and to verify our model. In addition a ¹H NMR based intermediate method is planed to prove if the so interpreted acyclic R5P is indeed covalently linked to the coenzyme. Interestingly, the only hydrogen bonding partner for the aldehyde oxygen is the cofactor N4´-amino group. A hydrogen bonding interaction with Gln428 has to be excluded as the

ThDP

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interatomic distance of less than 2 Å clearly predicts repulsion. This is again a difference to the EcTK-R5P complex for which a stabilizing interaction with His473 was reported (Asztalos et al., 2007)(Fig.

61). In analogy to the mechanistic analysis performed for E4P we superimposed the hTK-R5P complex with the DHEThDP intermediate (Fig. c.)) to visualize the carboligation reaction in this case to form the S7P-ThDP intermediate. The alignment reveals that the reacting atoms, R5P C1 and DHEThDP C2α, accomondate closely (< 0.5 Å) but in an unfavorable orientation. Larger positional rearrangements of the entire R5P molecule would be necessary to engage the corresponding positions in the S7P-ThDP intermediate. It seems to be more reasonable that the carboligation reaction proceeds immediately after furansoe ring opening of cyclic R5P. In consequence, our structural data indicate that for both native acceptor substrates the reactant state with the DHEThDP intermediate is probably transient.

Fig. 66: Proposed Models for ring opening of the acceptor substrate R5P in hTK. A general, schematic mechanism for acid-base catalized ring opening of R5P (α furanose) is depicted on top. Ring opening requires protonation of the ring oxygen (O4) and deprotonation of the anomeric hydroxyl group (O1). In Model 1 His37 would act as base and acid to perform a cyclic proton transfer. For the alternative Model 2 an active site water molecule (W1) would protonate the ring oxygen and His37 would deprotonated the anomeric hydroxyl group. The direction of the proposed proton transfer models is highlighted by arrows. Putative hydrogen bonding interaction (dashed blue lines) and selected distances for each model are shown.

The conversion of cyclic α furanose form of R5P into the acyclic aldehyde form (100 s^-1 at 40 °C) is not rate-limiting for the overall reaction (EcTK kcat = 40-50 s^-1)(Asztalos, 2007 b; Sprenger et al., 1995)(hTK kcat = 6-19 s ^-1)(Mitschke et al., 2010; Schenk et al., 1998) but for the carboligation with the DHEThDP intermediate (approx. 530 s^-1 for EcTK). Two models (Fig. 66) are feasible how hTK could accelerates the ring opening reaction of cyclic R5P. In Model 1, which is very reasonable in terms of bond length and proton transfer geometry, His37 would act simultaneous as Brønstedt base and acid to enable a cyclic proton relay.

Alternatively, in Model 2 a water molecule could act as proton donor while His37 could deprotonate the anomeric hydroxyl group. As already mentioned a deviation from coplanarity of 11.7° was observed for R5P bound to the active site of hTK that fits quite well to the 10-14° distortion reported for R5P bound to EcTK (Asztalos et al., 2007). Furanose rings are conformationally inhomogeneous in

O4

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solution and exist in multiples envelope- and twist conformers. Given that the energy barrier between those conformers is relatively small a rapid interconversion of conformers is typical in solution (Levitt and Warshel, 1978). Hence, it is reasonable to assume that such deviations from coplanarity result from ordinary flexibility of the furanose ring. In contrast the small molecule x-ray structure of R5P present in C2 endo conformation (Koziol and Lis, 1991) shows a perfectly coplanar arrangement (0.2°

deviation). Since no absolute energies for a coplanar and the observed distorted envelope conformation of enzyme bound R5P are yet available the small distortion can´t be addressed as a driving force for ring opening.