Alkaline phosphatase metal substitution

Alkaline phosphatase, 2,774 mechanism, 6,612 metal substitution, 6,611 structure, 6,611 zinc, 5.1006 6,610 Alkanes... 

Cobalt has recently been used as an ESR active substitute in zinc metalloenzymes. Whilst liquid helium temperatures may be needed and theoretical aspects of the spectra are not yet as well understood, cobalt has two important advantages over copper as a metal substitute, namely that many cobalt derivatives show some enzymic activity (e.g. cobalt in carbonic anhydrase, alkaline phosphatase and superoxide dismutase) and that g values and hyperfine splitting are more sensitive to ligand environment, particularly when low spin. ESR data have been reported for cobalt substituted thermolysin, carboxypeptidase A, procarboxypeptidase A and alkaline phosphatase [51]. These are all high spin complexes. Cobalt carbonic anhydrase has been prepared and reacted with cyanide [52]. In... 

Numerous metalloenzymes have the ability to remain functional even after the metal, which presumably is present at their active center, has been replaced by another metal (13). Thus in zinc deficiency, if the apoenzyme is synthesized, as has been observed in the case of . coli alkaline phosphatase (13), then other metals which might have accumulated or are normally within the cell could substitute for zinc and generate an active enzyme. Although this is a possibility in the case of microorganisms, it certainly does not appear to be true in the case of experimental animals and man, in that the apoenzymes of alkaline phosphatase, carbonic anhydrase, carboxypeptidase, alcohol dehydrogenase, and de-oxythymidine kinase do not accumulate in zinc-deficient tissues. Thus, one may conclude that a deficiency of zinc does specifically aflFect the activities of zinc-dependent enzymes in sensitive tissues. 

Much attention has been paid to metal-substituted alkaline phosphatases, notably Co" d-d spectra), Mn" (ESR) and Cd ( Cd NMR). The apoenzyme may be prepared by use of ammonium sulfate to remove zinc. After about five days the apoenzyme may be isolated having less than 3% of the original zinc. Furthermore, the apoenzyme is uncontaminated by chelating agents, which show a tendency to bind to the apoenzyme. A range of metalloalkaline phosphatases may be prepared from the apoenzyme. The binding of cadmium at three separate sites can be confirmed by the use of " Cd NMR, which shows " three separate resonances at 153, 72 and 3 p.p.m. in the phosphorylated dimer Cd"6AP. When all three sites are occupied by Cd , the enzyme has a very low turnover, at least 10 times slower than the native Zn" enzyme. This slow turnover number has made the Cd" enzyme particularly useful in NMR studies. 

The four zinc ions in alkaline phosphatase have also been replaced by other bivalent metal ions to probe the metal-binding site. The apodimer binds four cobalt(ii) ions, two Co binding to each of two different types of binding sites. Magnesium activates the cobalt-substituted enzyme, as it does the native zinc... 

The substitution of cobalt for the native zinc ions of alkaline phosphatase results in an active enzyme with distinctive optical properties, generated by the interaction of cobalt with the ligands of the protein. These properties may be employed to investigate the modes of binding of cobalt to the enzyme and also serve in a remarkable fashion to distinguish the catalytically essential metal atoms from those which play only a structural role. 

Fig. 2 demonstrates effects of various metal ions on the activity of the kinase reaction. Compared with the control in lane (i) metal ions are essential for kinase activity. Mg , showing the strongest effect, however, can be substituted by other divalent cations such as Mn, Cu " and Zn", in contrast Ca" " is ineffective in replacing Mg ". The addition of alkaline phosphatase had no effect on B875 phosphorylation. 

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