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The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase

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Abstract

Pyruvate kinase catalyses the final step of the glycolytic pathway in central energy metabolism. The monomeric structure comprises three domains: a catalytic TIM-barrel, a regulatory domain involved in allosteric activation, and a lid domain that encloses the substrates. The lid domain is thought to close over the TIM-barrel domain forming contacts with the substrates to promote catalysis and may be involved in stabilising the activated state when the allosteric activator is bound. However, it remains unknown whether the lid domain is essential for pyruvate kinase catalytic or regulatory function. To address this, we removed the lid domain of Escherichia coli pyruvate kinase type 1 (PKTIM+Reg) using protein engineering. Biochemical analyses demonstrate that, despite the absence of key catalytic residues in the lid domain, PKTIM+Reg retains a low level of catalytic activity and has a reduced binding affinity for the substrate phosphoenolpyruvate. The enzyme retains allosteric activation, but the regulatory profile of the enzyme is changed relative to the wild-type enzyme. Analytical ultracentrifugation and small-angle X-ray scattering data show that, beyond the loss of the lid domain, the PKTIM+Reg structure is not significantly altered and is consistent with the wild-type tetramer that is assembled through interactions at the TIM and regulatory domains. Our results highlight the contribution of the lid domain for facilitating pyruvate kinase catalysis and regulation, which could aid in the development of small molecule inhibitors for pyruvate kinase and related lid-regulated enzymes.

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Acknowledgements

This research was undertaken in part using the SAXS/WAXS beamline at the Australian Synchrotron, part of ANSTO, and we thank the staff of SAXS/WAXS beamlines for their assistance in data collection. DC acknowledges the University of Canterbury Doctoral Scholarship for funding support. We acknowledge Sarah Atkinson for help during project setup and Jackie Healy for technical support.

Funding

This work is supported by the New Zealand Royal Society Marsden Fund (contract UOC1013) and the US Army Research Office under contract/grant number W911NF-11–1-0481.

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Author notes

  1. Elena Sugrue and David Coombes contributed equally to this work.

Authors and Affiliations

  1. Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, PO Box 4800, Christchurch, 8140, New Zealand

    Elena Sugrue, David Coombes, David Wood, Tong Zhu, Katherine A. Donovan & Renwick C. J. Dobson

  2. MRC-University of Glasgow Centre for Virus Research, Glasgow, G61 1QH, UK

    Elena Sugrue

  3. Department of Cancer Biology, Dana–Farber Cancer Institute, Boston, MA, 02215, USA

    Katherine A. Donovan

  4. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA

    Katherine A. Donovan

  5. Biol21 Molecular Science and Biotechnology Institute, Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC, 3010, Australia

    Renwick C. J. Dobson

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  1. Elena Sugrue
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Contributions

ES, KAD, and RCJD conceived the project. ES conducted the experiments. ES, DW, and DC analysed and interpreted the data. RCJD secured funding for the work. ES, DW, and DC drafted the manuscript. All authors read and edited the manuscript.

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Correspondence to Renwick C. J. Dobson.

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Special Issue: Analytical Ultracentrifugation 2019

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Sugrue, E., Coombes, D., Wood, D. et al. The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase. Eur Biophys J 49, 761–772 (2020). https://doi.org/10.1007/s00249-020-01466-5

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