Apparent negative co-operativity and substrate inhibition in overexpressed glutamate dehydrogenase from Escherichia coli

Sharkey, Michael A.; Engel, Paul C.

doi:10.1111/j.1574-6968.2008.01086.x

Cited by 22 publications

(30 citation statements)

References 25 publications

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“…The K m values for the substrates and products of purified GDH of E. coli as measured by six different groups differed considerably: the ranges of K m values were 0.2 to 6.0 mM for 2-oxoglutarate, 1 to 36 mM for ammonium, and 12 to 83 M for NADPH (21). The K m values for the substrates and products of purified GDH of S. Typhimurium lie in the respective ranges (60).…”

Section: Glutamate Dehydrogenasementioning

confidence: 99%

“…Purified GDH showed substantial substrate inhibition with 2-oxoglutarate (Ͼ10 mM) and glutamate (Ͼ250 mM) (21). Purified GDH showed a pH optimum at 8.0 for the reductive amination reaction (20,21), while that of S. Typhimurium showed a pH optimum at 8.6 (55). Interestingly, GDH has been shown to be phosphorylated at a histidine residue in vitro (62) and in vivo (63) in the exponential growth phase.…”

Section: Glutamate Dehydrogenasementioning

confidence: 99%

“…The K m values for the substrates and products of purified GDH of S. Typhimurium lie in the respective ranges (60). Purified GDH showed substantial substrate inhibition with 2-oxoglutarate (Ͼ10 mM) and glutamate (Ͼ250 mM) (21). Purified GDH showed a pH optimum at 8.0 for the reductive amination reaction (20,21), while that of S. Typhimurium showed a pH optimum at 8.6 (55).…”

Section: Glutamate Dehydrogenasementioning

confidence: 99%

“…Purified GDH of E. coli was heat stable (19,20) and remained active when stored for several months at room temperature, while it was inactivated by freezing; potassium protected against freezing damage (19). In other hands, purified GDH was found to lose activity within minutes at room temperature (21). Purified GDH displayed an unusual resistance to high concentrations of the protein denaturant urea (20) and guanidine HCl (19).…”

Section: Glutamate Dehydrogenasementioning

confidence: 99%

“…Purified GDH has a relatively high K m value for ammonium (ϳ1 mM) (19,21), whereas purified GS has a 10-foldlower K m for ammonium, i.e., ϳ0.1 mM (22)(23)(24). Therefore, the received view is that the "cheap and low-affinity" enzyme GDH takes care of nitrogen assimilation during growth in high-ammonium and low-carbon/energy media, while the "free energy-expensive and high-affinity" enzyme system GS-GOGAT functions during growth in low-ammonium and high-carbon/energy media.…”

Section: Gs-gogat Pathwaymentioning

confidence: 99%

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Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

Heeswijk¹,

Westerhoff

Boogerd³

2013

Microbiol Mol Biol Rev

226

246

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SUMMARY We present a comprehensive overview of the hierarchical network of intracellular processes revolving around central nitrogen metabolism in Escherichia coli . The hierarchy intertwines transport, metabolism, signaling leading to posttranslational modification, and transcription. The protein components of the network include an ammonium transporter (AmtB), a glutamine transporter (GlnHPQ), two ammonium assimilation pathways (glutamine synthetase [GS]-glutamate synthase [glutamine 2-oxoglutarate amidotransferase {GOGAT}] and glutamate dehydrogenase [GDH]), the two bifunctional enzymes adenylyl transferase/adenylyl-removing enzyme (ATase) and uridylyl transferase/uridylyl-removing enzyme (UTase), the two trimeric signal transduction proteins (GlnB and GlnK), the two-component regulatory system composed of the histidine protein kinase nitrogen regulator II (NRII) and the response nitrogen regulator I (NRI), three global transcriptional regulators called nitrogen assimilation control (Nac) protein, leucine-responsive regulatory protein (Lrp), and cyclic AMP (cAMP) receptor protein (Crp), the glutaminases, and the nitrogen-phosphotransferase system. First, the structural and molecular knowledge on these proteins is reviewed. Thereafter, the activities of the components as they engage together in transport, metabolism, signal transduction, and transcription and their regulation are discussed. Next, old and new molecular data and physiological data are put into a common perspective on integral cellular functioning, especially with the aim of resolving counterintuitive or paradoxical processes featured in nitrogen assimilation. Finally, we articulate what still remains to be discovered and what general lessons can be learned from the vast amounts of data that are available now.

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Section: Glutamate Dehydrogenasementioning

confidence: 99%

Section: Glutamate Dehydrogenasementioning

confidence: 99%

Section: Glutamate Dehydrogenasementioning

confidence: 99%

Section: Glutamate Dehydrogenasementioning

confidence: 99%

Section: Gs-gogat Pathwaymentioning

confidence: 99%

See 3 more Smart Citations

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

Heeswijk¹,

Westerhoff

Boogerd³

2013

Microbiol Mol Biol Rev

226

246

View full text Add to dashboard Cite

show abstract

Progress in Scaling up and Streamlining a Nanoconfined, Enzyme‐Catalyzed Electrochemical Nicotinamide Recycling System for Biocatalytic Synthesis

2020

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An electrochemically driven nicotinamide recycling system, referred to as the ‘electrochemical leaf’ has unique attributes that may suit it to the small‐scale industrial synthesis of high‐value chemicals. A complete enzyme cascade can be immobilized within the channels of a nanoporous electrode, allowing complex reactions to be energized, controlled and monitored continuously in real time. The electrode is easily prepared by depositing commercially available indium tin oxide (ITO) nanoparticles on a Ti support, resulting in a network of nanopores into which enzymes enter and bind. One of the enzymes is the photosynthetic flavoenzyme, ferredoxin NADP+ reductase (FNR), which catalyzes the quasi‐reversible electrochemical recycling of NADP(H) and serves as the transducer. The second enzyme is any NADP(H)‐dependent dehydrogenase of choice, and further enzymes can be added to build elaborate cascades that are driven in either oxidation or reduction directions through the rapid recycling of NADP(H) within the pores. In this Article, we describe the measurement of key enzyme/cofactor parameters and an essentially linear scale‐up from an analytical scale 4 mL reactor with a 14 cm2 electrode to a 500 mL reactor with a 500 cm2 electrode. We discuss the advantages (energization, continuous monitoring that can be linked to a computer, natural enzyme immobilization, low costs of electrodes and low cofactor requirements) and challenges to be addressed (optimizing minimal use of enzyme applied to the electrode).

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Modular coenzyme specificity: A domain‐swopped chimera of glutamate dehydrogenase

Sharkey¹,

Engel²

2009

Proteins

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Domain-swopped chimeras of the glutamate dehydrogenases from Clostridium symbiosum (CsGDH) (NAD(+)-specific) and Escherichia coli (EcGDH) (NADP(+)-specific) have been produced, with the aim of testing the localization of determinants of coenzyme specificity. An active chimera consisting of the substrate-binding domain (Domain I) of CsGDH and the coenzyme-binding domain (Domain II) of EcGDH has been purified to homogeneity, and a thorough kinetic analysis has been carried out. Results indicate that selectivity for the phosphorylated coenzyme does indeed reside solely in Domain II; the chimera utilizes NAD(+) at 0.8% of the rate observed with NADP(+), similar to the 0.5% ratio for EcGDH. Positive cooperativity toward L-glutamate, characteristic of CsGDH, has been retained with Domain I. An unforeseen feature of this chimera, however, is that, although glutamate cooperativity occurs only at higher pH values in the parent CsGDH, the chimeric protein shows it over the full pH range explored. Also surprising is that the chimera is capable of catalysing severalfold higher reaction rates (V(max)) in both directions than either of the parent enzymes from which it is constructed.

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Apparent negative co-operativity and substrate inhibition in overexpressed glutamate dehydrogenase from Escherichia coli

Cited by 22 publications

References 25 publications

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

Progress in Scaling up and Streamlining a Nanoconfined, Enzyme‐Catalyzed Electrochemical Nicotinamide Recycling System for Biocatalytic Synthesis

Modular coenzyme specificity: A domain‐swopped chimera of glutamate dehydrogenase

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