scholarly journals Effects of zinc and other metal ions on the stability and activity of Escherichia coli alkaline phosphatase

1971 ◽  
Vol 124 (1) ◽  
pp. 25-30 ◽  
Author(s):  
C. N. A. Trotman ◽  
C. Greenwood
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Metal Ions ◽  
Phosphatase Activity ◽  
Structural Stability ◽  
Zinc Content ◽  
Sedimentation Velocity ◽  
Reducing Agent ◽  
Deficient Mutant ◽  
The Stability

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.

scholarly journals Escherichia coli alkaline phosphatase. Kinetic studies with the tetrameric enzyme

1972 ◽  
Vol 126 (5) ◽  
pp. 1081-1090 ◽  
Author(s):  
S. E. Halford ◽  
M. J. Schlesinger ◽  
H. Gutfreund
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Steady State ◽  
Analytical Ultracentrifugation ◽  
Kinetic Studies ◽  
Product Formation ◽  
E Coli ◽  
Enzyme Subunits ◽  
Self Association ◽  
The Stability

1. The stability of the tetrameric form of Escherichia coli alkaline phosphatase was examined by analytical ultracentrifugation. 2. The stopped-flow technique was used to study the hydrolysis of nitrophenyl phosphates by the alkaline phosphatase tetramer at pH7.5 and 8.3. In both cases transient product formation was observed before the steady state was attained. Both transients consisted of the liberation of 1mol of nitrophenol/2mol of enzyme subunits within the dead-time of the apparatus. The steady-state rates were identical with those observed with the dimer under the same conditions. 3. The binding of 2-hydroxy-5-nitrobenzyl phosphonate to the alkaline phosphatase tetramer was studied by the temperature-jump technique. The self-association of two dimers to form the tetramer is linked to a conformation change within the dimer. This accounts for the differences between the transient phases in the reactions of the dimer and the tetramer with substrate. 4. Addition of Pi to the alkaline phosphatase tetramer caused it to dissociate into dimers. The tetramer is unable to bind this ligand. It is suggested that the tetramer undergoes a compulsory dissociation before the completion of its first turnover with substrate. 5. On the basis of these findings a mechanism is proposed for the involvement of the alkaline phosphatase tetramer in the physiology of E. coli.

scholarly journals Constitutive Mutations in the Escherichia coli AraC Protein

2009 ◽  
Vol 191 (8) ◽  
pp. 2668-2674 ◽  
Author(s):  
Stephanie Dirla ◽  
John Yeh-Heng Chien ◽  
Robert Schleif
Keyword(s):  
Escherichia Coli ◽  
Circular Dichroism ◽  
Structural Stability ◽  
Dimerization Domain ◽  
Helix Bundle ◽  
The Core ◽  
Switch Mechanism ◽  
The Stability ◽  
Circular Dichroïsm

ABSTRACT The Escherichia coli AraC protein represses and induces the araBAD operon in response to the absence or presence of l-arabinose. Constitutive mutations in the AraC gene no longer require the presence of l-arabinose to convert AraC from its repressing to its inducing state. Such mutations were isolated directly by virtue of their constitutivity or by their resistance to the nonmetabolizable arabinose analog, d-fucose. The majority of the constitutive mutations lie within the same residues of the N-terminal regulatory arm of AraC. Two, however, were found in the core of the dimerization domain. As predicted by the light switch mechanism of AraC, constitutive mutations increase the susceptibility of the N-terminal arms to digestion by trypsin or chymotrypsin, suggesting that these mutations weaken or disrupt the arm structure required for repression by AraC. Fluorescence, circular dichroism, and cysteine reactivity measurements show that the constitutive mutations in the core of the dimerization domain lead to a weakening of the support for the arms and reduce the stability of the minus-arabinose arm structure. These mutations also weaken the interaction between the two-helix bundle and the β-barrel subdomains of the dimerization domain and reduce the structural stability of the β-barrels.

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