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Sub-ångström-resolution crystallography reveals physical distortions that enhance reactivity of a covalent enzymatic intermediate

Abstract

It is recognized widely that enzymes promote reactions by providing a pathway that proceeds through a transition state of lower energy. In principle, further rate enhancements could be achieved if intermediates are prevented from relaxing to their lowest energy state, and thereby reduce the barrier to the subsequent transition state. Here, we report sub-ångström-resolution crystal structures of genuine covalent reaction intermediates of transketolase. These structures reveal a pronounced out-of-plane distortion of over 20° for the covalent bond that links cofactor and substrate, and a specific elongation of the scissile substrate carbon–carbon bond (d > 1.6 Å). To achieve these distortions, the protein's conformation appears to prevent relaxation of a substrate–cofactor intermediate. The results implicate a reduced barrier to the subsequent step that is consistent with an intermediate of raised energy and leads to a more efficient overall process.

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Figure 1: TK reactions.
Figure 2: Structural evidence for physically distorted covalent intermediates in human TK.
Figure 3: Origin of strain in the covalent X5P–ThDP conjugate bound to human TK.
Figure 4: Principles of enzyme catalysis.

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Acknowledgements

We thank G. M. Sheldrick for providing a pre-release version of software SHELXL-2012. We are grateful for synchrotron beam time and local support at the ESRF Grenoble, France, and at the BESSY Berlin, Germany. This work was supported through grants of the Fonds der Chemischen Industrie (K.T.), Forschergruppe 1296 funded by the Deutsche Forschungsgemeinschaft (K.T.) and the Sonderforschungsbereich 860 funded by the Deutsche Forschungsgemeinschaft (R.F.).

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Authors

Contributions

K.T. conceived and supervised the project; S.L. expressed, purified and crystallized the enzyme; S.L. and P.N. collected data sets at ESFR Grenoble and BESSY Berlin; S.L. refined structures; P.N. conducted final refinement rounds and e.s.d. values using the software SHELXL-2012; S.L. conducted CD measurements; K.M.E. and F.L. synthesized the thiamin analogue; K.T., S.L., P.N., K.M.E., F.L., R.F. and R.K. discussed the data. R.K. and K.T. co-wrote the paper with input from all other authors. S.L. and P.N. contributed equally to this study.

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Correspondence to Kai Tittmann.

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Lüdtke, S., Neumann, P., Erixon, K. et al. Sub-ångström-resolution crystallography reveals physical distortions that enhance reactivity of a covalent enzymatic intermediate. Nature Chem 5, 762–767 (2013). https://doi.org/10.1038/nchem.1728

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