Altering of the metal specificity of Escherichia coli alkaline phosphatase.

Metal specificity is correlated with two crucial active site residues in Escherichia coli alkaline phosphatase.

Escherichia coli alkaline phosphatase exhibits maximal activity when Zn(2+) fills the M1 and M2 metal sites and Mg(2+) fills the M3 metal site. When other metals replace the zinc and magnesium, the catalytic efficiency is reduced by more than 5000-fold. Alkaline phosphatases from organisms such as Thermotoga maritima and Bacillus subtilis require cobalt for maximal activity and function poorly ...

Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme.

The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54¿ omitted¿760 Da. On the alignment of ...

Cobalt(III), a probe of metal binding sites of Escherichia coli alkaline phosphatase.

To facilitate the study of individual metal binding sites of polymeric metalloproteins, conversion of exchange-labile Co(II) in E. coli alkaline phosphatase (EC 3.1.3.1) to exchange-inert Co(III) was examined. Oxidation of Co(II) alkaline phosphatase with hydrogen peroxide results in a single absorption maximum at 530 nm and loss both of the characteristic electron paramagnetic signal and of en...

Escherichia coli alkaline phosphatase. Metal binding, protein conformation, and quaternary structure.

Simplified and accurate nonradioactive polynucleotide gene probe assay for identification of enterotoxigenic Escherichia coli.

The present study describes a colony hybridization setup for identification of enterotoxigenic Escherichia coli obviating the need for advanced equipment and radioactive isotopes. With a modest laboratory arrangement, polynucleotide gene probes were produced in large quantities. The probes were labeled with digoxigenin and, after hybridization, detected with an antidigoxigenin alkaline phosphat...