The control of pyruvate kinases of Escherichia coli: Further studies of the enzyme activated by ribose-5-phosphate

1978 ◽  
Vol 56 (6) ◽  
pp. 647-653 ◽  
Author(s):  
John S. Mort ◽  
B. D. Sanwal
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Kinase ◽  
Quaternary Structure ◽  
Native Enzyme ◽  
Open Structure ◽  
Allosteric Inhibitor ◽  
Pyruvate Kinases ◽  
Allosteric Activator

The pyruvate kinase activated by ribose-5-phosphate from Escherichia coli has been purified to homogeneity, taking advantage of the stabilization of the enzyme by its inhibitor phosphate and by thiol reagents. The native enzyme has a tetrameric quaternary structure which, while prone to dissociation under many conditions, remains intact in the presence of the above reagents.The enzyme was found to reactivate on dilution out of 8 M urea. Interestingly, the recovery of activity is greatly increased by phosphate, an allosteric inhibitor, but markedly reduced by the allosteric activator, ribose-5-phosphate, implying that it is harder for the enzyme to refold to a 'relaxed state.' Proteolysis studies indicate a more open structure in the presence of the activator.

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

Biochimie ◽  
2010 ◽  
Vol 92 (1) ◽  
pp. 116-120 ◽  
Author(s):  
Tong Zhu ◽  
Michael F. Bailey ◽  
Lauren M. Angley ◽  
Timothy F. Cooper ◽  
Renwick C.J. Dobson
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Kinase ◽  
Quaternary Structure

scholarly journals Transcriptional and Biochemical Characterization of Cytosolic Pyruvate Kinases in Arabidopsis thaliana

Plants ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 353
Author(s):  
Sabine Wulfert ◽  
Sören Schilasky ◽  
Stephan Krueger
Keyword(s):  
Arabidopsis Thaliana ◽  
Pyruvate Kinase ◽  
Expression Pattern ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Quaternary Structure ◽  
Tricarboxylic Acid Cycle ◽  
Yellow Fluorescent Protein ◽  
Allosteric Effectors ◽  
Pyruvate Kinases

Glycolysis is a central catabolic pathway in every living organism with an essential role in carbohydrate breakdown and ATP synthesis, thereby providing pyruvate to the tricarboxylic acid cycle (TCA cycle). The cytosolic pyruvate kinase (cPK) represents a key glycolytic enzyme by catalyzing phosphate transfer from phosphoenolpyruvate (PEP) to ADP for the synthesis of ATP. Besides its important functions in cellular energy homeostasis, the activity of cytosolic pyruvate kinase underlies tight regulation, for instance by allosteric effectors, that impact stability of its quaternary structure. We determined five cytosol-localized pyruvate kinases, out of the fourteen putative pyruvate kinase genes encoded by the Arabidopsis thaliana genome, by investigation of phylogeny and localization of yellow fluorescent protein (YFP) fusion proteins. Analysis of promoter β-glucuronidase (GUS) reporter lines revealed an isoform-specific expression pattern for the five enzymes, subject to plant tissue and developmental stage. Investigation of the heterologously expressed and purified cytosolic pyruvate kinases revealed that these enzymes are differentially regulated by metabolites, such as citrate, fructose-1,6-bisphosphate (FBP) and ATP. In addition, measured in vitro enzyme activities suggest that pyruvate kinase subunit complexes consisting of cPK2/3 and cPK4/5 isoforms, respectively, bear regulatory properties. In summary, our study indicates that the five identified cytosolic pyruvate kinase isoforms adjust the carbohydrate flux through the glycolytic pathway in Arabidopsis thaliana, by distinct regulatory qualities, such as individual expression pattern as well as dissimilar responsiveness to allosteric effectors and enzyme subgroup association.

Detection of Escherichia coli using luminometer with pyruvate kinase

2021 ◽  
Author(s):  
Huaiqun Liu ◽  
Yuanyuan Shen ◽  
Peng Zhao ◽  
Yuxin Liu
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Kinase

Pyruvate kinase from the liver and kidney of Arapaima gigas

1978 ◽  
Vol 56 (4) ◽  
pp. 852-859 ◽  
Author(s):  
H. Guderley ◽  
J. H. A. Fields ◽  
J. M. Cardenas ◽  
P. W. Hochachka
Keyword(s):  
Pyruvate Kinase ◽  
Arapaima Gigas ◽  
Liver And Kidney ◽  
Low Levels ◽  
Pyruvate Kinases ◽  
Regulatory Properties ◽  
Partial Reversal

Pyruvate kinases from the kidney and liver of the osteoglossid Arapaima gigas were partially purified and characterized kinetically. The two enzymes have different elect rophoretic mobilities at pH 7.0, and while they share some qualitative similarities they show quantitative differences in their catalytic and regulatory properties. Both enzymes are activated by fructose 1.6-bisphosphate and inhibited by low levels of alanine and MgATP. The liver isozyme shows hyperbolic phosphoenolpyruvate binding, with a K1 for alanine inhibition of 0.7 mM and a K1 for MgATP inhibition of 1.0 mM. In contrast, the kidney isozyme shows cooperative phosphoenolpyruvate binding, which is accentuated at low levels of ADP. MgATP inhibition does not increase the cooperativity and shows an apparent K1 of 1.68 mM. The inhibition of alanine leads to considerable increases in the cooperativity and is effective at 1 mM and lower levels. Fructose 1.6-bisphosphate completely reverses the inhibition by alanine for both isozymes, while only leading to a partial reversal of the MgATP inhibition. These regulatory properties of both the kidney and the liver isozymes suit them for function in tissues which undergo both glycolysis and gluconeogenesis.

Two forms of pyruvate kinase in Escherichia coli a comparison of chemical and molecular properties

1979 ◽  
Vol 570 (2) ◽  
pp. 248-258 ◽  
Author(s):  
Giovanna Valentini ◽  
Paolo Iadarola ◽  
Babu Lal Somani ◽  
Massimo Malcovati
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Kinase ◽  
Molecular Properties

scholarly journals The catalase-peroxidase of Synechococcus PCC 7942: purification, nucleotide sequence analysis and expression in Escherichia coli

1996 ◽  
Vol 316 (1) ◽  
pp. 251-257 ◽  
Author(s):  
Michinori MUTSUDA ◽  
Takahiro ISHIKAWA ◽  
Toru TAKEDA ◽  
Shigeru SHIGEOKA
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Molecular Mass ◽  
Peroxidase Activity ◽  
Catalase Activity ◽  
Native Enzyme ◽  
Nucleotide Sequence Analysis ◽  
Synechococcus Pcc 7942 ◽  
Catalase Peroxidase ◽  
Pcc 7942

Synechococcus PCC 7942, a cyanobacterium, possesses catalase–peroxidase as the sole hydrogen peroxide-scavenging system. The enzyme has been purified to electrophoretic homogenenity from the cells. The native enzyme had a molecular mass of 150 kDa and was composed of two identical subunits of molecular mass 79 kDa. The apparent Km value of the catalase activity for H2O2 was 4.2±0.27 mM and the kcat value was 2.6×104 s-1. The enzyme contained high catalase activity and an appreciable peroxidase activity with o-dianisidine and pyrogallol. The catalase activity was not inhibited by 3-amino-1,2,4-triazole but by KCN and NaN3 (apparent Ki values 19.3±0.84 and 20.2±0.95 μM respectively). The enzyme showed an absorption spectrum of typical protohaem and contained one protohaem molecule per dimer. The gene encoding catalase–peroxidase was cloned from the chromosomal DNA of Synechococcus PCC 7942. A 2160 bp open reading frame (ORF), coding a catalase–peroxidase of 720 amino acid residues (approx. 79.9 kDa), was observed. The deduced amino acid sequence coincided with that of the N-terminus of the purified enzyme and showed a remarkable similarity to those of a family of catalase–peroxidases of prokaryotic cells. Escherichia coli BL21(DE3)plysS, harbouring a recombinant plasmid containing the catalase–peroxidase gene, produced a large amount of proteins that co-migrated on SDS/PAGE with the native enzyme. The recombinant enzyme showed the same ratio of catalase activity to peroxidase activity with o-dianisidine and the same Km for H2O2 as the native enzyme.

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