Alkaline phosphatase of Escherichia coli.  Composition

Simpson, Robert T.; Vallée, Bert L.; Tait, G. H.

doi:10.1021/bi00852a028

Cited by 93 publications

(51 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…MATERIALS AND METHODS Alkaline phosphatase was released by osmotic shock from the periplasmic space of E. coli cells treated with EDTA (2) Abbreviation: EPR, electron paramagnetic resonance. * To whom correspondence should be addressed.…”

Section: T)mentioning

confidence: 99%

“…The importance of zinc in both catalysis and structure stabilization of Escherichia coli alkaline phosphatase has long been recognized (1), but the presence of stoichiometric amounts of magnesium (1,2) has been largely ignored; hence, its role has remained both unappreciated and undefined. This study confirms the presence of stoichiometric quantities of magnesium in alkaline phosphatase and further demonstrates that this metal serves to regulate the mode of binding of zinc and increases enzymatic activities over and above those attained in the presence of zinc alone.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Role of magnesium in Escherichia coli alkaline phosphatase.

Anderson¹,

Bosron²,

Kennedy³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Alkaline phosphatase of E. coli, isolated by procedures which do not alter its intrinsic metal content, contains 1.3 I 0.3 g-atom of magnesium and 4.0 4 0.2 g-atom of zinc per molecule of molecular weight 89,000. Magnesium, the role of which has been unappreciated, significantly affects the function and structure of alkaline phosphatase containing either 2 or 4 g-atom of zinc per mole. Magnesium does not'activate the apoenzyme but increases the activity of the enzyme containing. 2 g-atom of zinc 4.4-fold and that of the enzyme containing 4 g-atom 1.2-fold. The results obtained with enzyme in which cobalt is substituted for zinc are analogous. Moreover, the absorption and electron paramagnetic resonance spectra of cobalt phosphatases reveal the effects of magnesium on cobalt coordination geometry. Addition of magnesium changes the spectral characteristics of the apoenzyme reconstituted with 2 g-atom of cobalt from predominantly octahedral to 4-or 5-coordinate geometry. These two classes of cobalt binding sites have been associated with catalysis and structure stabilization, respectively. Therefore, magnesium controls the occupancy of the catalytic and structural binding sites and modulates the resultant enzymatic activity. Hydrogen-tritium exchange was employed to determine the effects of magnesium on the conformational stability of phosphatase. Magnesium stabilizes the dynamic structural properties, both of apophosphatase and of enzyme containing 2 g-atom of zinc, which is further stabilized by 2 more zinc atoms. The role of magnesium and other metal ions in regulatory processes, only now beginning to be explored fully, will likely emerge as an important avenue for achievement of regulatory effects in metalloenzymes.The importance of zinc in both catalysis and structure stabilization of Escherichia coli alkaline phosphatase has long been recognized (1), but the presence of stoichiometric amounts of magnesium (1, 2) has been largely ignored; hence, its role has remained both unappreciated and undefined. This study confirms the presence of stoichiometric quantities of magnesium in alkaline phosphatase and further demonstrates that this metal serves to regulate the mode of binding of zinc and increases enzymatic activities over and above those attained in the presence of zinc alone. The response of the absorption and electron paramagnetic resonance (EPR) spectra of cobalt alkaline phosphatase to magnesium delineates details of its regulatory roles in cobaltand indirectly zinc-binding and in the consequent modulations of activity (3, t).MATERIALS AND METHODS Alkaline phosphatase was released by osmotic shock from the periplasmic space of E. coli cells treated with EDTA (2) Abbreviation: EPR, electron paramagnetic resonance.

show abstract

Section: T)mentioning

confidence: 99%

mentioning

confidence: 99%

Role of magnesium in Escherichia coli alkaline phosphatase.

Anderson¹,

Bosron²,

Kennedy³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…This is reflected in the several bond as a short loop (nine intervening residues). The other was amino acid compositions that have been reported (6,29,30). formed by two peptides that were severed by performic oxiThese data, particularly those of Simpson et al (29), are in exdation and separated by gel filtration.…”

mentioning

confidence: 97%

“…The other was amino acid compositions that have been reported (6,29,30). formed by two peptides that were severed by performic oxiThese data, particularly those of Simpson et al (29), are in exdation and separated by gel filtration. Their location in CN II cellent agreement with the composition obtained from the seand CN lb was determined from their amino acid composition.…”

mentioning

confidence: 97%

Amino acid sequence of Escherichia coli alkaline phosphatase.

Bradshaw¹,

Cancedda²,

Ericsson³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

162

View full text Add to dashboard Cite

The complete amino acid sequence of the Escherichia coli alkaline phosphatase subunit [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1, isozyme 3] has been determined. The monomer contains 449 amino acid residues in a single unglycosylated polypeptide chain having a calculated Mr of47,029. Isozyme 1 has an additional arginine residue at the NH2 terminus that presumably results from variability in processing of precursor molecules. Sequence data were obtained from both manual and automatic Edman degradation ofthe tryptic and cyanogen bromide peptides, as well as other peptides derived therefrom. The two disulfide bonds were determined from analyses of the appropriate peptic peptides. This structure confirms earlier reports of the sequence surrounding the active-site serine and both the NH2-and COOH-terminal cyanogen bromide fragments. A secondary structure prediction places nearly half the residues in a-helical segments that have 13% and 16%, respectively, in fl-strand and fl-turn orientations.

show abstract

“…Studies on Escherichia coli alkaline phosphatase (ECAP) have shown the importance of the divalent metal ions: Mg 2+ does not directly participate in the mechanism but is important for stabilization of the enzyme, whereas the two Zn 2+ ions are directly involved in catalysis (21,22). The Mg 2+ can affect the structure and function of molecular chromophores (23) as well as of many enzymes (24,25).…”

Section: +mentioning

confidence: 99%