The quaternary structure of pyruvate kinase type 1 from Escherichia coli at low nanomolar concentrations

Zhu, Tong; Bailey, Michael F.; Angley, Lauren M.; Cooper, Tim F.; Dobson, Renwick C. J.

doi:10.1016/j.biochi.2009.09.016

Cited by 17 publications

(17 citation statements)

References 17 publications

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“…PEP carboxylase is a tetrameric enzyme, compromised of a four identical catalytic units 37 . The same is true of PykF 32 . A likely reason for the evolution of multimeric enzymes is to enable ultrasensitivity, to substrate 38–42 , to feedback inhibitors in biosynthesis 43,44 , or, as shown here, to feed forward activation in central carbon metabolism.…”

Section: Discussionmentioning

confidence: 67%

“…15), and thus PEP accumulating when glycolytic flux decreases, as has been observed experimentally in E. coli and other organisms 30,31 . Like PEP carboxylase, the pyruvate kinase isozyme which is expressed in the presence of glucose (PykF) is activated by FBP 32 . We anticipate that, in the presence of physiological metabolite concentrations, PykF will also respond ultrasensitively to FBP, rendering PEP accumulation a robust physiological response to glucose removal.…”

Section: Discussionmentioning

confidence: 99%

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Ultrasensitive regulation of anapleurosis via allosteric activation of PEP carboxylase

Amador‐Noguez

Reaves

et al. 2012

Nat Chem Biol

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Anapleurosis is the filling of the TCA cycle with four-carbon units. The common substrate for both anapleurosis and glucose phosphorylation in bacteria is the terminal glycolytic metabolite, phosphoenolpyruvate (PEP). Here we show that E. coli quickly and almost completely turns off PEP consumption upon glucose removal. The resulting build-up of PEP is used to quickly import glucose if it becomes re-available. The switch-like termination of anapleurosis results from depletion of fructose-1,6-bisphosphate (FBP), an ultrasensitive allosteric activator of PEP carboxylase. E. coli expressing an FBP-insensitive point mutant of PEP carboxylase grow normally on steady glucose. However, they fail to build-up PEP upon glucose removal, grow poorly on oscillating glucose, and suffer from futile cycling at the PEP node on gluconeogenic substrates. Thus, bacterial central carbon metabolism is intrinsically programmed with ultrasensitive allosteric regulation to enable rapid adaptation to changing environmental conditions.

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Section: Discussionmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Ultrasensitive regulation of anapleurosis via allosteric activation of PEP carboxylase

Amador‐Noguez

Reaves

et al. 2012

Nat Chem Biol

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“…3PG was modeled as an inhibitor for isocitrate lyase/malate synthase [29, 33]. FDP was modeled as an activator for pyruvate kinase [29, 39] and PEP carboxylase [29, 40]. Pyruvate was modeled as an inhibitor for pyruvate dehydrogenase [29, 41, 42] and as an activator for lactate dehydrogenase [43].…”

Section: Methodsmentioning

confidence: 99%

Toward a Genome Scale Sequence Specific Dynamic Model of Cell-Free Protein Synthesis inEscherichia coli

Horvath

Vilkhovoy²,

Wayman

et al. 2017

Preprint

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Cell-free protein expression systems have become widely used in systems and synthetic biology. In this study, we developed an ensemble of dynamic E. coli cell-free protein synthesis (CFPS) models. Model parameters were estimated from a training dataset for the cell-free production of a protein product, chloramphenicol acetyltransferase (CAT). The dataset consisted of measurements of glucose, organic acids, energy species, amino acids, and CAT. The ensemble accurately predicted these measurements, especially those of the central carbon metabolism. We then used the trained model to evaluate the optimality of protein production. CAT was produced with an energy efficiency of 12%, suggesting that the process could be further optimized. Reaction group knockouts showed that protein productivity and the metabolism as a whole depend most on oxidative phosphorylation and glycolysis and gluconeogenesis. Amino acid biosynthesis is also important for productivity, while the overflow metabolism and TCA cycle affect the overall system state. In addition, the translation rate is shown to be more important to productivity than the transcription rate. Finally, CAT production was robust to allosteric control, as was most of the network, with the exception of the organic acids in central carbon metabolism. This study is the first to use kinetic modeling to predict dynamic protein production in a cell-free E. coli system, and should provide a foundation for genome scale, dynamic modeling of cell-free E. coli protein synthesis.Introduction 1 Cell-free protein expression has become a widely used research tool in 2 systems and synthetic biology, and a promising technology for personalized 3 point of use biotechnology [1]. Cell-free systems offer many advantages for 4 the study, manipulation and modeling of metabolism compared to in vivo 5 processes. Central amongst these, is direct access to metabolites and the 6 biosynthetic machinery without the interference of a cell wall, or complica-7 tions associated with cell growth. This allows us to interrogate (and po-8 tentially manipulate) the chemical microenvironment while the biosynthetic 9 machinery is operating, potentially at a fine time resolution. Cell-free pro-10 tein synthesis (CFPS) systems are arguably the most prominent examples 11 of cell-free systems used today [2]. However, CFPS is not new; CFPS in 12 crude E. coli extracts has been used since the 1960s to explore fundamental 13 biological mechanisms. For example, Matthaei and Nirenberg used E. coli 14 cell-free extract in ground-breaking experiments to decipher the sequencing 15 of the genetic code [3, 4]. Spirin and coworkers later improved protein pro-16 duction in cell free extracts by continuously exchanging reactants and prod-17 ucts; however, while these extracts could run for tens of hours, they could 18 only synthesize a single product and were energy limited [5]. More recently, 19 energy and cofactor regeneration in CFPS has been significantly improved; 20 for example ATP can be regenerated using substrate level pho...

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“…The purified protein (38 M in 200 mM ammonium acetate, pH 6.9) was introduced into the glass capillary of the mixing device at a flow rate of 1 l/min, whereas the D 2 O was introduced into the outer metal capillary at a flow rate of 3 l/min using Harvard 11ϩ infusion pumps. Where appropriate, the enzyme was preincubated with 2 mM fructose 1,6-bisphosphate (10 times the K D of 0.2 mM (30,(35)(36)(37)) to ensure the saturation of the allosteric binding sites. The fluids followed a laminar flow pattern until they reached the notch in the glass capillary at which point they were combined and mixed.…”

Section: Methodsmentioning

confidence: 99%

Conformational Dynamics and Allostery in Pyruvate Kinase

Donovan

Zhu

Liuni

et al. 2016

Journal of Biological Chemistry

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Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the "domain rotation model" suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the "rigid body reorientation model" proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogendeuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an ␣-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the ␤-strands within the (␤/␣) 8 -barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site.

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The quaternary structure of pyruvate kinase type 1 from Escherichia coli at low nanomolar concentrations

Cited by 17 publications

References 17 publications

Ultrasensitive regulation of anapleurosis via allosteric activation of PEP carboxylase

Ultrasensitive regulation of anapleurosis via allosteric activation of PEP carboxylase

Toward a Genome Scale Sequence Specific Dynamic Model of Cell-Free Protein Synthesis inEscherichia coli

Conformational Dynamics and Allostery in Pyruvate Kinase

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