The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase
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ORIGINAL ARTICLE
The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase Elena Sugrue1,2 · David Coombes1 · David Wood1 · Tong Zhu1 · Katherine A. Donovan1,3,4 · Renwick C. J. Dobson1,5 Received: 2 June 2020 / Revised: 24 August 2020 / Accepted: 14 September 2020 © European Biophysical Societies’ Association 2020
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 P KTIM+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. Keywords Pyruvate kinase · Analytical ultracentrifugation · Protein engineering · Glycolysis · Enzyme evolution · Enzyme kinetics
Introduction Glycolysis is the central energy-generating pathway in most organisms (Van Schaftingen 1993). The pathway catalyses the breakdown of glucose into pyruvate and this is coupled to energy generation via the synthesis of reduced nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate
Elena Sugrue and David Coombes contributed equally to this work. Special Issue: Analytical Ultracentrifugation 2019 Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00249-020-01466-5) contains supplementary material, which is available to authorized users. * Renwick C. J. Dobson [email protected] Extended author information available on the last page of the article
(ATP) that are then used to drive further biosynthetic processes (Ainscow and Brand 1999; Xie and Wang 1996). The final step of glycolysis is catalysed by the enzyme pyruvate kinase and this reaction is o
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