Escherichia coli alkaline phosphatase

Cambella and Antia [385] determined phosphonates in seawater by fractionation of the total phosphorus. The seawater sample was divided into two aliquots. The first was analysed for total phosphorus by the nitrate oxidation method capable of breaking down phosphonates, phosphate esters, nucleotides, and polyphosphates. The second aliquot was added to a suspension of bacterial (Escherichia coli) alkaline phosphatase enzyme, incubated for 2h at 37 °C and subjected to hot acid hydrolysis for 1 h. The resultant hot acid-enzyme sample was assayed for molybdate reactive phosphate which was estimated as the sum of enzyme hydrolysable phosphate and acid hydrolysable... 

Applebury, M. L., and Coleman, J. E. Escherichia coli alkaline phosphatase. Metal binding, protein conformation, and quaternary structure. J. Biol. Chem. 244, 308—318 (1969). 

The Escherichia coli alkaline phosphatase (li coli AP) is the most extensively studied phosphatase, and perhaps the most studied two-metal ion catalyst.68,91 95 The AP-catalyzed reaction proceeds via an intermediate in which a serine residue (Ser-102 in E. coli AP) is phosphorylated. Thus, the stereochemical outcome of the overall reaction is retention. The hydrolysis of the intermediate by water to produce inorganic phosphate competes with phosphoryl transfer to other acceptors such as alcohols or nucleophilic buffers if such are present in solution. The rate-limiting step... 

Lakshmi and Balasubramanian (1980) showed the presence of a new multiple form of arylsulfohydrolase B in human and monkey brain. Arylsulfohydrolase B, can be separated by DEAE-cellulose chromatography (Mathew and Balasubramanian, 1984). The B, form totally binds to Sephadex G-200 and was not eluted with 1.0 M NaCl, 0.5 M glucose, 0.5 M glucose plus 0.5 M NaCl, 0.5 M KSCN, 1 M urea, or 1% Triton X-100. The treatment of arylsulfohydrolase B with Escherichia coli alkaline phosphatase results in the formation of a less acidic form, presumably due to dephosphorylation. The dephosphorylated form does not bind to DEAE-cellulose. Inorganic phosphate and serine phosphate but not mannose 6-phosphate can inhibit this dephosphorylation. The kinetic properties of the phosphorylated and dephosphorylated arylsulfohydrolase are quite similar. The possibility that arylsulfohydrolase B is a dephosphorylated form of B, has been ruled out by the significant differences between substrate concentration and activity curves of these enzymes. 

At this point it should be noted that not all slow steps in protein folding are prolyl isomerizations. The very slow refolding of large proteins is often limited in rate by other events, such as slow conformational rearrangements, domain-pairing reactions, or subunit associations. An extreme example is provided by the Escherichia coli alkaline phosphatase. This protein requires days to complete folding, but, clearly, this very slow refolding reaction is not related to prolyl isomerization (Dirnbach et al., 1999). 

A. Escherichia coli Alkaline Phosphatase and Related Enzymes... 

Shan, Y., McKelvie, I.D. and Hart, B.T. (1993) Characterisation of immobilised Escherichia coli alkaline phosphatase reactors in flow injection analysis. Analytical Chemistry 65, 3053-3060. 

It is noteworthy that three His, Glu, Asp or Cys residues provide zinc ligands for all known enzyme catalytic zinc sites [ 30], Water is the fourth ligand and histidine is by far the most frequent amino acid among the catalytic site residues. Three histidines are found in human carbonic anhydrases 1 and II, p-lactamase, the DD-carboxypeptidase of Streptomyces albus G, adenosine deaminase and astacin [30]. Two histidines are characteristic of bovine carboxypeptidases A and B, thermolysin and Escherichia coli alkaline phosphatase... 

Halford, S. E. (1972). Escherichia coli alkaline phosphatase. Biochemical Journal, 126,311-22. 

In the case of Escherichia coli alkaline phosphatase, a dimeric zinc metal-loenzyme where a total of six metal ions (three per monomer) are involved directly or indirectly in providing the enzyme with maximal catalytic activity and structural stability, Cd NMR, in conjunction with and NMR methods, were instrumental in separating out the function of each class of metal binding sites. Perhaps most importantly, these studies revealed the chemical basis for negative coopera-tivity that had been reported for this enzyme under metal deficient conditions. Also noteworthy was the fact that these NMR studies preceeded the availability of the X-ray crystal structure. 

Escherichia coli alkaline phosphatase, a dimeric zinc metalloenzyme ( 95,500 Da), binds 2 Zn ions and 1 Mg ion per monomer and functions in the non-specific hydrolysis of phosphate monoester. NMR (mi labeled histidine biosyntheti-cally incorporated into AP in conjunction with substrate NMR and Cd NMR methods were used for the assigmnent of the three Cd resonances to specific sites per monomer and their role in substrate binding. A full account of these studies can be found in the following references [6,25,80,82,201],... 

Angelini, S., Moreno, R., Gouffi, K. et al. (2001) Export of Thermus thermophilus alkaline phosphatase via the twin-arginine translocation pathway in Escherichia coli. FEBS Letters, 506 (2), 103-107. 

Isolation of alkaline phosphatase from Escherichia coli in which 85% of the proline residues were replaced by 3,4-dehydro-proline affected the heat lability and ultraviolet spectrum of the protein but the important criteria of catalytic function such as the and were unaltered (12). Massive replacement of methionine by selenomethionine in the 0-galactosidase of E. coli also failed to influence the catalytic activity. Canavanine facilely replaced arginine in the alkaline phosphatase of this bacterium at least 13 and perhaps 20 to 22 arginyl residues were substituted. This replacement by canavanine caused subunit accumulation since the altered subunits did not dimerize to yield the active enzyme (21). Nevertheless, these workers stated "There was also formed, however, a significant amount of enzymatically active protein in which most arginine residues had been replaced by canavanine." An earlier study in which either 7-azatryptophan or tryptazan replaced tryptophan resulted in active protein comparable to the native enzyme (14). 

Izutani, K., Nakata, A., Shinagawa, H. Kawamata, J. (1980) Forward mutation assay for screening carcinogens by alkaline phosphatase constitutive mutations in Escherichia coli K-12. BikenJ.,13,69-75... 

Later, it was found that alkaline phosphatase was also released by osmotic shock E. coli were exposed to 0.5 M sucrose containing dilute tris-HCl buffer and EDTA, and then the centrifuged cells were rapidly dispersed in the shock medium of cold water or cold 5 X 10 4 M MgCl2. Although the cells were 80% viable with the latter case, almost all of the enzyme was released (9, 10). Other evidence indicates that the only important structural effect of EDTA is to increase the permeability of the cell wall (11, 12). Escherichia coli grow normally in the... 

Escherichia coli can he adapted to grow on PPi as the sole source of phosphorus in this situation the cells are dependent upon intracellular inorganic pyrophosphatase activity to make Pi available. To avoid possible confusion from the inducible alkaline phosphatase of E. coli, which also has pyrophosphatase activity (see Chapter 17, by Reid and Wilson, this volume), all mutant isolation studies began with an E coli strain unable to synthesize this inducible protein. 

M3. Malamy, M. H., and Horecker, B. L., Release of alkaline phosphatase from cells of Escherichia coli upon lysozyme spheroplast formation. Biochemistry 3, 1889-1893 (1964). 

M6. Manson, L. A., Pelmont, J., Yapo, A., Roche, C., and Nisman, B., The biosynthesis of alkaline phosphatase with a particulate fraction of Escherichia coli. The mechanism of inhibition by inorganic phosphate. Biochem. J. 96, 215-225 (1965). 

M20. Milstein, C., The amino acid sequence around the reactive serine residue in alkaline phosphatase from Escherichia coli. Biochem. J. 92, 410-421 (1964). 

Sohlesinger, M. J., The reversible dissociation of the alkaline phosphatase of Escherichia coli. II. Properties of the subunit. J. Biol. Chem. 240, 4293-4298 (1965). 

WIO. Wilson, I. B., Dayan, J., and Cyr, K., Some properties of alkaline phosphatase from Escherichia coli. Transphosphorylation. J. Biol. Chem. 239, 4182-4185 (1964). 

---