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

Sugrue, E. , Coombes, D., Wood, D., Zhu, T., Donovan, K. A. and Dobson, R. C.J. (2020) The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase. European Biophysics Journal, 49, pp. 761-772. (doi: 10.1007/s00249-020-01466-5) (PMID:32978636)

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.

• Item Type: Articles
• Additional Information: 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.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Sugrue, Dr Elena
• Authors: Sugrue, E., Coombes, D., Wood, D., Zhu, T., Donovan, K. A., and Dobson, R. C.J.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity; College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
• Journal Name: European Biophysics Journal
• Publisher: Springer
• ISSN: 0175-7571
• ISSN (Online): 1432-1017
• Published Online: 25 September 2020
• Copyright Holders: Copyright © 2020 European Biophysical Societies' Association
• First Published: First published in European Biophysics Journal 49:761–772
• Publisher Policy: Reproduced in accordance with the publisher copyright policy