Bond Lengths Analysis of the Donor-ThDP Intermediates

TKs cleave two strong C-C bonds either to enable donor release (C2-C2α bond) or donor cleavage (C2α-C3α bond). In order to facilitate C-C bond cleavage proton abstraction from either O2α (donor release) or O3α (donor cleavage) forming presumably transient alkoxids is required (Fig. 42). The product of those cleavage reactions is a C2α cabanion in resonance with its enamine component or the

N1´

N4´

S7P-ThDP F6P-ThDP

N4´

N1´

N4´

N1´

X5P-ThDP

C2´

N3´

C5´

C4´

C6

C6´

C7´

89

cofactor C2 carbanion in resonance with a C2 carbene. This electronic stabilization can be considered as a thermodynamic driving force for C-C bond cleavage in both cases. Furthermore, ionization of other sugar-derived OH-groups like O4α can potentially occur. The products of those C-C-bond cleavage reactions are isolated carbanions like the C3α carbanion depicted in the middle scheme of Fig. 42. Isolated carbanions lacking any potential stabilization are unstable and the equilibrium of the reaction lies consequently on the side of the uncleaved state.

Fig. 42: Reversible C-C bond cleavage of donor-ThDP intermediates. Chemical structure of donor-ThDP adduct including possible C-C bond cleavage mechanism via the formation of carbanions. Scissile C-C bonds are highlighted in red. Brønsted bases (B1, B2 and B3) are labeled. For explaination see text.

Analysis of all three native donor substrate-intermediates revealed very similar tendencies. The scissile C2α-C3α and C2-C2α bonds are significantly elongated (Tab. 5, Tab. 13) indicating that they are already relaxed by physical stress or electronic effects potentially facilitating the subsequent cleavage reactions. The presence of these long bonds was verified for the X5P- and F6P-ThDP intermediate by estimation of standard deviation using a pre-release version of SHELXL-11 (Sheldrick, 2008) which was kindly provided by Prof. George Sheldrick. Here, it is important to consider that the C2-C2α

R2

90

bond, which connects an aromatic ring carbon with an exocylcic carbon, should be slightly shorter than a standard C-C bond of 154 pm (Allen et al., 1987).

Tab. 5: Selected C-C-bond lengths of hTK in complex with X5P-ThDP, F6P-ThDP (refined with SHELXL-11) and S7P-ThDP (refined with PHENIX). For X5P- and F6P-ThDP and estimation of standard deviation was calculated.

(* C2α-C3α and C2-C2α bonds were not included for calculation)

In cases of the C2α-C3α bond physical stress could be generated by two attractive “poles”, the active site residues positioning the substrate derived phosphate moiety and the strained C2-C2α bond which itself is pulled into a planar arrangement. However, the hydrogen bonding network around the phosphate moiety is not rigid and should allow smaller positional changes arguing against a tight

“anchor” function for this group. Another origin for the enlarged C2α-C3α bond could be the presence of a small fraction DHEThDP intermediate and an aldose (cleavage products). This possibility has to be excluded because acceptor sugars adopt sp² hybridization at C1 which is not supported by the electron density map. It is further conceivable that the cofactor pulles binding electrons into the thiazolium ring (electron sink) thereby causing the observed prolongated and C-C bonds. A bond prolongation of 8 pm (e.g. 154 to 162 pm) would dramatically decrease the bond dissociation energy by more than 30 kcal/mol, from 90 kcal/mol to less than 60 kcal/mol (Zavitsas, 2003). Importantly, all stated, speculative mechanisms have to be validated by computational or independent biophysical methods.

While elongated C-C bonds could be observed for sterically compressed, small-molecule structures (Kaupp and Boy, 1997; Maslak et al., 1995; Schreiner et al., 2011; Toda, 2000) there is to our knowledge no structural information available for an enzymatic reaction intermediate possessing such characteristics. Notably, Chabriére and co-workers (Chabriere et al., 2001) succeded to trap a covalent reaction intermediate (pdb-code:1kek) for the ThDP-dependent enzyme pyruvate:ferridoxin oxidoreductase with exceptionally long C-C bonds (C2-C2α bond 175-195 pm) connecting cofactor and substrate-derived part. However, the authors could prove a fractional radical character of those long bonds (“one-electron” bond) which is compatible with knowledge from small-molecule cation radicals (Bellvile et al., 1985). Since neither cofactor- nor substrate-centered radical species could so far be detected at any reaction step of TK this source for the elongated C2α-C3α bond can probably be excuded. The involvement of radicals is further unlikely due to the lack of any redox-active cofactor like flavins or iron-sulfur clusters in TK which is a prerequisite for ThDP-mediated radical

C2α-C3α bond (pm)

C2α-C2α bond (pm)

avarage of all sugar derived C-C bonds * (pm)

range of all sugar derived C-C bonds * (pm)

X5P-ThDP 162.5 ± 1.5 152.3 ± 1.2 151.8 148.0-155.1

F6P-ThDP 155.2 ± 1.7 160.4 ± 1.5 153.1 149.2-156.2

S7P-ThDP 162 161 152.2 151-153

91

biochemistry (Tittmann, 2009). However, X-band electron paramagmnetic resonance spectroscopic measurements of enzyme bound donor-ThDP intermediates are currently in preparation to prove the presence or absence of radical species.

Furthermore the C2α-O2α and C3α-O3α bonds are slightly but significantly shorter than all other C-O single bonds of the intermediate suggesting that a small fraction of O2α and O3α might be deprotonated and present as alkoxids. However, for the S7P-ThDP intermediate the C3α-O3α is not shortened.

Tab. 6: Selected C-O-bond lengths of hTK in complex with X5P-ThDP, F6P-ThDP (refined with SHELXL-11) and S7P-ThDP (refined with Phenix). For X5P- and F6P-ThDP and estimation of standard deviation was calculated.

(* C2α-C3α and C2-C2α bonds were not included for calculation)

After nucleophilic attack of the C2 carbanion to the keto carbon of the donor O2α is indeed first present as an alkoxide. Since no potential general acid is located in a suitable orientation the functional group acting as proton donor is very likely the 4´-amino group of the cofactor itself (APH⁺ form) which thereby changes its protonation state from APH⁺ to IP form (Nemeria et al., 2009).

While the determined bond lengths between C4 and C5 of 137-139 pm suggest a predominant double bond character, both bonds that C2 is involved in are significantly enlarged (C2-N3 136–

140 pm instead of 132 pm, C2-S1 172–173 pm instead of 167 pm, Tab. 13).

The bond lengths in the six-membered IP moiety have similar values relative to those observed for small molecule structures of ThDP present as AP form (Pletcher et al., 1979). Solely the C5´-C6´ and C6´-N1´ bonds are slightly elongated. A correlation of this enlarged bond with catalytic competence must yet remain open.

3.4.2.6. Structural Evidence for the Presence of Angular Strain in Both Aromatic Ring