Transcription of the Escherichia coli fliC gene is regulated by metal ions.

Measurement of the ultraviolet circular dichroism of apo-(alkaline phosphatase) in urea solutions showed substantial denaturation in 3m-urea. A zinc-deficient mutant alkaline phosphatase behaved similarly. The stability of the enzyme in 6m-urea was followed as a function of its zinc content and was found to be dependent on the first two of the four zinc atoms bound by apoenzyme. Phosphatase activity was mostly dependent on a second pair of zinc atoms. Mn2+, Co2+, Cu2+ or Cd2+ also restored structural stability. Sedimentation-velocity and -equilibrium experiments revealed that dissociation of the dimer accompanied apoenzyme denaturation in urea concentrations of 1m or higher, without treatment with disulphide-reducing agent.

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Investigation of the nature of the two metal-binding sites in 5-aminolaevulinic acid dehydratase from Escherichia coli

Biochemical Journal ◽

10.1042/bj3000373 ◽

1994 ◽

Vol 300 (2) ◽

pp. 373-381 ◽

Cited By ~ 24

Author(s):

P Spencer ◽

P M Jordan

Keyword(s):

Escherichia Coli ◽

Metal Ions ◽

Metal Binding ◽

Binding Sites ◽

Metal Ion ◽

Protein Fluorescence ◽

Metal Binding Sites ◽

Aminolaevulinic Acid ◽

Acid Dehydratase ◽

Aminolaevulinic Acid Dehydratase

Two distinct metal-binding sites, termed alpha and beta, have been characterized in 5-aminolaevulinic acid dehydratase from Escherichia coli. The alpha-site binds a Zn2+ ion that is essential for catalytic activity. This site can also utilize other metal ions able to function as a Lewis acid in the reaction mechanism, such as Mg2+ or Co2+. The beta-site is exclusively a transition-metal-ion-binding site thought to be involved in protein conformation, although a metal bound at this site only appears to be essential for activity if Mg2+ is to be bound at the alpha-site. The alpha- and beta-sites may be distinguished from one another by their different abilities to bind divalent-metal ions at different pH values. The occupancy of the beta-site with Zn2+ results in a decrease of protein fluorescence at pH 6. Occupancy of the alpha- and beta-sites with Co2+ results in u.v.-visible spectral changes. Spectroscopic studies with Co2+ have tentatively identified three cysteine residues at the beta-site and one at the alpha-site. Reaction with N-ethyl[14C]maleimide preferentially labels cysteine-130 at the alpha-site when Co2+ occupies the beta-site.

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Subunit location of the intrinsic divalent metal ions in RNA polymerase from Escherichia coli

Biochemistry ◽

10.1021/bi00644a008 ◽

1977 ◽

Vol 16 (25) ◽

pp. 5449-5454 ◽

Cited By ~ 46

Author(s):

Cheng-Wen Wu ◽

Felicia Y. H. Wu ◽

David C. Speckhard

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Metal Ions ◽

Divalent Metal ◽

Divalent Metal Ions

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Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes' strain)

Biochemical Journal ◽

10.1042/bj1310833 ◽

1973 ◽

Vol 131 (4) ◽

pp. 833-841 ◽

Cited By ~ 67

Author(s):

Donald Stribling ◽

Richard N. Perham

Keyword(s):

Escherichia Coli ◽

Molecular Weight ◽

Metal Ions ◽

Gel Filtration ◽

Deae Cellulose ◽

Class Ii ◽

Class I ◽

E Coli ◽

Km Value ◽

Bivalent Metal Ions

Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca2+, Zn2+. It is a dimer with a molecular weight of approx. 70000, and the Km value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH4 in the presence of substrate. The Km value for fructose diphosphate is about 20μm (although the Lineweaver–Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed.

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