CTP synthetase from Escherichia coli: an improved purification procedure and characterization of hysteretic and enzyme concentration effects on kinetic properties

Biochemistry ◽  
1983 ◽  
Vol 22 (13) ◽  
pp. 3285-3292 ◽  
Author(s):  
Paul M. Anderson
Keyword(s):  
Escherichia Coli ◽  
Enzyme Concentration ◽  
Kinetic Properties ◽  
Purification Procedure ◽  
Ctp Synthetase ◽  
Concentration Effects

Comparison of the structural and kinetic properties of the cytochrome c nitrite reductases from Escherichia coli, Wolinella succinogenes, Sulfurospirillum deleyianum and Desulfovibrio desulfuricans

2006 ◽  
Vol 34 (1) ◽  
pp. 143-145 ◽  
Author(s):  
T.A. Clarke ◽  
A.M. Hemmings ◽  
B. Burlat ◽  
J.N. Butt ◽  
J.A. Cole ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Active Site ◽  
Desulfovibrio Desulfuricans ◽  
Amino Acid Sequences ◽  
Kinetic Properties ◽  
Wolinella Succinogenes ◽  
Nitrite Reductases ◽  
Sulphite Reductase

The recent crystallographic characterization of NrfAs from Sulfurospirillum deleyianum, Wolinella succinogenes, Escherichia coli and Desulfovibrio desulfuricans allows structurally conserved regions to be identified. Comparison of nitrite and sulphite reductase activities from different bacteria shows that the relative activities vary according to organism. By comparison of both amino acid sequences and structures, differences can be identified in the monomer–monomer interface and the active-site channel; these differences could be responsible for the observed variance in substrate activity and indicate that subtle changes in the NrfA structure may optimize the enzyme for different roles.

scholarly journals Function-Based Selection and Characterization of Base-Pair Polymorphisms in a Promoter of Escherichia coli RNA Polymerase-ς70

2001 ◽  
Vol 183 (9) ◽  
pp. 2866-2873 ◽  
Author(s):  
Jian Xu ◽  
Barbara C. McCabe ◽  
Gerald B. Koudelka
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Promoter Sequence ◽  
Kinetic Properties ◽  
Optimal Sequence ◽  
Sequence Requirements ◽  
Better Than

ABSTRACT We performed two sets of in vitro selections to dissect the role of the −10 base sequence in determining the rate and efficiency with which Escherichia coli RNA polymerase-ς70forms stable complexes with a promoter. We identified sequences that (i) rapidly form heparin-resistant complexes with RNA polymerase or (ii) form heparin-resistant complexes at very low RNA polymerase concentrations. The sequences selected under the two conditions differ from each other and from the consensus −10 sequence. The selected promoters have the expected enhanced binding and kinetic properties and are functionally better than the consensus promoter sequence in directing RNA synthesis in vitro. Detailed analysis of the selected promoter functions shows that each step in this multistep pathway may have different sequence requirements, meaning that the sequence of a strong promoter does not contain the optimal sequence for each step but instead is a compromise sequence that allows all steps to proceed with minimal constraint.

scholarly journals Kinetic Properties of Four Plasmid-Mediated AmpC β-Lactamases

2005 ◽  
Vol 49 (10) ◽  
pp. 4240-4246 ◽  
Author(s):  
Cédric Bauvois ◽  
Akiko Shimizu Ibuka ◽  
Almeida Celso ◽  
Jimena Alba ◽  
Yoshikazu Ishii ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Kinetic Parameters ◽  
Kinetic Properties ◽  
Kinetic Characterization ◽  
Heterologous Production ◽  
Periplasmic Space ◽  
E Coli ◽  
Class C ◽  
Susceptibility Patterns

ABSTRACT The heterologous production in Escherichia coli, the purification, and the kinetic characterization of four plasmid-encoded class C β-lactamases (ACT-1, MIR-1, CMY-2, and CMY-1) were performed. Except for their instability, these enzymes are very similar to the known chromosomally encoded AmpC β-lactamases. Their kinetic parameters did not show major differences from those obtained for the corresponding chromosomal enzymes. However, the Km values of CMY-2 for cefuroxime, cefotaxime, and oxacillin were significantly decreased compared to those of the chromosomal AmpC enzymes. Finally, the susceptibility patterns of different E. coli hosts producing a plasmid- or a chromosome-encoded class C enzyme toward β-lactam antibiotics are mainly due to the overproduction of the β-lactamase in the periplasmic space of the bacteria rather than to a specific catalytic profile of the plasmid-encoded β-lactamases.

Kinetic properties of 7α-hydroxysteroid dehydrogenase from Escherichia coli 080

1989 ◽  
Vol 35 (12) ◽  
pp. 1076-1080 ◽  
Author(s):  
V. Prabha ◽  
Meenakshi Gupta ◽  
K. G. Gupta
Keyword(s):  
Escherichia Coli ◽  
Bile Acids ◽  
Sodium Perchlorate ◽  
Tween 80 ◽  
Thiol Group ◽  
Hydroxysteroid Dehydrogenase ◽  
Enzyme Concentration ◽  
Tween 20 ◽  
Kinetic Properties ◽  
Potassium Oxalate

Results on the kinetics of 7α-hydroxysteroid dehydrogenase 7α-HSDH showed that this enzyme could oxidize all bile acids having an –OH group at the C-7 position. Lineweaver-Burk plots showed Michaelis constant (Km) values of 0.83 and 0.12 mM for cholic acid and chenodeoxycholic acid, respectively. The effect of enzyme concentration on the reaction velocity showed a constant increase in the enzyme activity with increase in enzyme-protein concentration. 7α-HSDH was activated by Na+, K+, Ca2+, and Mn2+ ions and by reducing agents having a thiol group (dithiothreitol, 2-mercaptoethanol). Co2+, Hg2+, Fe3+, Mg2+, Zn2+, Ba2+, and Cu2+ ions, chelating agents (potassium oxalate, heparin, EDTA), oxidizing agents (sodium perchlorate, sodium periodate, sodium persulphate), and detergents (Tween 20, Tween 40, Tween 80, Triton X-100, sodium lauryl sulphate) were inhibitory to 7α-HSDH activity.Key words: 7α-hydroxysteroid dehydrogenase, bile acids, NAD+, Escherichia coli 080, enzyme kinetics.

scholarly journals Isolation and Characterization of an Aldehyde Dehydrogenase Encoded by the aldB Gene of Escherichia coli

2005 ◽  
Vol 187 (3) ◽  
pp. 1067-1073 ◽  
Author(s):  
Kwok Ki Ho ◽  
Henry Weiner
Keyword(s):  
Escherichia Coli ◽  
Aldehyde Dehydrogenase ◽  
Kinetic Properties ◽  
Isolation And Characterization ◽  
Cell Extracts ◽  
Bacterial Enzyme ◽  
Terminal Amino ◽  
Mammalian Enzyme ◽  
In Cells

ABSTRACT An aldehyde dehydrogenase was detected in crude cell extracts of Escherichia coli DH5α. Growth studies indicated that the aldehyde dehydrogenase activity was growth phase dependent and increased in cells grown with ethanol. The N-terminal amino acid sequence of the purified enzyme identified the latter as an aldehyde dehydrogenase encoded by aldB, which was thought to play a role in the removal of aldehydes and alcohols in cells that were under stress. The purified enzyme showed an estimated molecular mass of 220 ± 8 kDa, consisting of four identical subunits, and preferred to use NADP and acetaldehyde. MgCl2 increased the activity of the NADP-dependent enzyme with various substrates. A comparison of the effect of Mg2+ ions on the bacterial enzyme with the effect of Mg2+ ions on human liver mitochondrial aldehyde dehydrogenase revealed that the bacterial enzyme shared kinetic properties with the mammalian enzyme. An R197E mutant of the bacterial enzyme appeared to retain very little NADP-dependent activity on acetaldehyde.

scholarly journals Escherichia coli Malic Enzymes: Two Isoforms with Substantial Differences in Kinetic Properties, Metabolic Regulation, and Structure

2007 ◽  
Vol 189 (16) ◽  
pp. 5937-5946 ◽  
Author(s):  
Federico P. Bologna ◽  
Carlos S. Andreo ◽  
María F. Drincovich
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Metabolic Regulation ◽  
Oxidative Decarboxylation ◽  
Kinetic Properties ◽  
Oligomeric State ◽  
E Coli ◽  
Divalent Metal Ion ◽  
High Degree

ABSTRACT Malic enzymes (MEs) catalyze the oxidative decarboxylation of malate in the presence of a divalent metal ion. In eukaryotes, well-conserved cytoplasmic, mitochondrial, and plastidic MEs have been characterized. On the other hand, distinct groups can be detected among prokaryotic MEs, which are more diverse in structure and less well characterized than their eukaryotic counterparts. In Escherichia coli, two genes with a high degree of homology to ME can be detected: sfcA and maeB. MaeB possesses a multimodular structure: the N-terminal extension shows homology to ME, while the C-terminal extension shows homology to phosphotransacetylases (PTAs). In the present work, a detailed characterization of the products of E. coli sfcA and maeB was performed. The results indicate that the two MEs exhibit relevant kinetic, regulatory, and structural differences. SfcA is a NAD(P) ME, while MaeB is a NADP-specific ME highly regulated by key metabolites. Characterization of truncated versions of MaeB indicated that the PTA domain is not essential for the ME reaction. Nevertheless, truncated MaeB without the PTA domain loses most of its metabolic ME modulation and its native oligomeric state. Thus, the association of the two structural domains in MaeB seems to facilitate metabolic control of the enzyme. Although the PTA domain in MaeB is highly similar to the domains of proteins with PTA activity, MaeB and its PTA domain do not exhibit PTA activity. Determination of the distinct properties of recombinant products of sfcA and maeB performed in the present work will help to clarify the roles of MEs in prokaryotic metabolism.

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