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Active site electronic structure and dynamics during metalloenzyme catalysis

Nature Structural Biology volume 10, pages 98–103 (2003)Cite this article

Abstract

Zinc-dependent enzymes play important roles in many cellular processes. Assignment of their reaction mechanisms is often a subject of debate because the zinc ion is silent in several spectroscopic techniques. We have combined time-resolved X-ray absorption spectroscopy, pre-steady state kinetics and computational quantum chemistry to study the active site zinc ion of bacterial alcohol dehydrogenase during single substrate turnover. We detect a series of alternations in the coordination number and structure of the catalytic zinc ion with concomitant changes in metal–ligand bond distances. These structural changes are reflected in the effective charge of the metal ion. The present work emphasizes the flexibility of catalytic zinc sites during catalysis and provides novel mechanistic insights into alcohol dehydrogenase catalysis.

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Figure 1: The catalytic site in the TbADH–NADP+ complex (PDB entry 1YKF).
Figure 2: Time-resolved freeze-quenched XAS of TbADH during a single catalytic cycle.
Figure 3: Real-time changes in the effective charge of the catalytic zinc ion during TbADH catalysis.
Figure 4: Proposed structural events around the catalytic zinc ion in TbADH within the context of the reaction mechanism of the oxidation of secondary alcohols.

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Acknowledgements

We thank M. Sullivan from the NSLS beam line X9B, K. Zhang and S. Stepanov from the APS the BioCat beamline and G. Kafri and E. Yavin from the Weizmann Institute for their help and technical advice. The authors are grateful to M.W. Makinen for critically reading this manuscript. This work was supported by the Israeli Science Foundation (I.S.) and by the Minerva Foundation (J.M.L.M.).

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Authors and Affiliations

  1. Department of Structural Biology, The Weizmann Institute of Science, Rehovot, 76100, Israel

    Oded Kleifeld & Irit Sagi

  2. Department of Physics, Yeshiva University, New York, 10016, New York, USA

    Anatoly Frenkel

  3. Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, 76100, Israel

    Jan M.L. Martin

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Correspondence to Irit Sagi.

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Kleifeld, O., Frenkel, A., Martin, J. et al. Active site electronic structure and dynamics during metalloenzyme catalysis. Nat Struct Mol Biol 10, 98–103 (2003). https://doi.org/10.1038/nsb889

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