Subunits alkaline phosphatase

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was also formed, however, a significant amount of enzymatically active protein in which most arginine residues had been replaced by canavanine."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). 

FIGURE 22. The active site of alkaline phosphatase (above) and an allosteric kinetic switch mechanism (below) for the regulatory function of the Mg + ions in controlling the conformation of the nonequivalent subunits (square and circle). Reprinted with permission from Reference 214. Copyright 2005 American Chemical Society... 

The alkaline phosphatase of E. coli is a dimer of 449-residue subunits which requires Zn2+, is allo-sterically activated by Mg2+, and has a pH optimum above 8.667/708 711 At a pH of 4, incubation of the enzyme with inorganic phosphate leads to formation of a phosphoenzyme. Using 32P-labeled phosphate, it was established that the phosphate becomes attached in ester linkages to serine 102. The same active site sequence Asp-Ser-Ala is found in mammalian alkaline phosphatases. These results, as well as the stereochemical arguments given in Section 2, suggest a double-displacement mechanism of Eq. 12-38 ... 

It has recently been reported (45) that when E. coli cells were suspended in 0.05 N HC1 subunits of the enzyme were quantitatively released into the medium. Subsequent reassociation and reactivation of these subunits provided an initial cell free extract that contained alkaline phosphatase which was 30% pure. 

Subunits do not form a precipitate with antiphosphatase antibody however, there appear to be some antigenic determinates common to both subunits and active enzyme since subunits interfere with the precipitation of alkaline phosphatase. The alkaline phosphatase-antibody complex has 70% of the original enzymic activity as a suspension in solution. Therefore, the antibody does not bind to the active site of alkaline phosphatase, but it can still differentiate between monomers and dimers. 

Rothman and Byrne 46) have used tryptic digestion to determine whether the subunits of alkaline phosphatase are identical. Since trypsin specifically cleaves at lysyl and arginyl residues, there will be as many peptides formed as there are arginine and lysine residues if the mono-... 

On the other hand, alkaline phosphatase may have two equivalent active sites which are coupled so that, normally, only one can operate at a time. This seems an attractive alternative for an enzyme consisting of two identical subunits. In a preliminary paper, Lazdunski et al. (125) report the covalent incorporation of two phosphates into the zinc enzyme as well as the cobalt enzyme, at >H<4. At these low pH values, the free enzyme generally loses its metal ions and dissociates into monomeres (109). However, if these results are corroborated after the performance of proper controls, and if both phosphates are linked to specific amino acid residues in the enzyme, conditions may have been found for the uncoupling of active sites in alkaline phosphatase. 

Alkaline phosphatase160-164 is a dimeric zinc metalloenzym composed of two identical subunits. The number of zinc atoms per protein molecule varies in different preparations. However, only two seem to be required for catalytic activity. The molecular weight of the monomer has been reported to be 42.000 so the natural dimer would be twice that value. Alkaline phosphatase is a phosphorylating enzyme and has 760 residues per dimer. 

Alkaline phosphatases form a well-known class of proteins that perform quite interesting and complicated reactions. As previously reported, Zn enzymes, like carboxypeptidases, thermolysin, and carbonic anhydrases, consist of only one Zn atom per active center. Most of the alkaline phosphatases consist of two 96-kDa subunits, each containing two Zn and one Mg ion. The alkaline phosphatase from E. coli has been crystallized and described in full detail [4], and a mechanism has been proposed. Several enzymes in this category have been mentioned in recent years, some of them also containing different metal ions, such as iron and zinc, as in the purple acid phosphatase [5], It is likely that the detailed structure and mechanism of many more examples of enzymes that remove or add phosphate groups to proteins will become available in the next decade. 

In general there are three phosphatase families alkaline, acid, and protein phosphatases. Alkaline phosphatases are typically dimers that contain three metal ions per subunit and have a pH optimum pH above 8. Acid phosphatases exhibit an optimum pH<7 and are usually divided into three classes low molecular weight acid phosphatases (<20 kDa), high molecular weight acid phosphatases (50-60 kDa), and purple acid phosphatases (which contain an Fe-Fe or Fe-Zn center at the active site). Phosphatases specific for I-l-P appear to be most similar (in kinetic characteristics but not in mechanism) to the alkaline phosphatases, but their structures define a superfamily that also includes inositol polyphosphate 1-phosphatase, fructose 1, 6-bisphosphatase, and Hal2. The members of this superfamily share a common structural core of 5 a-helices and 11 (3-strands. Many are Li+-sensitive (York et al., 1995), and more recent structures of archaeal IMPase proteins suggest the Li+ -sensitivity is related to the disposition of a flexible loop near the active site (Stieglitz et al., 2002). 

The alkaline phosphatase of E. coli is a dimer of 449-residue subunits which requires Zro , is allo-sterically activated by Mg +, and has a pH optimum above At a pH of 4, incubation of the... 

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). 

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). 

Metalloenzyme-catalyzed phosphoric ester hydrolysis can be illustrated by alkaline phosphatase, by far the most-investigated enzyme of this class. The protein is a dimer of 94 kDa containing two zinc(II) and one magnesium(II) ions per monomer, and catalyzes, rather unspecifically, the hydrolysis of a variety of phosphate monoesters as well as transphosphorylation reactions. The x-ray structure at 2.8 A resolution obtained on a derivative in which all the native metal ions were replaced by cadmium(II) reveals three metals in each subunit. 

An interesting solution is to color code the beads and the vessel in which the reaction is done.204 if there were eight subunits, for example, each one could be partitioned into different containers with different color caps. If each subunit were attached to a different color bead, one bead of each color could be added to each color-coded vessel. When the next subunit is attached, the compounds formed can be sorted individually by cap and by color. This process can be contained as each new subunit is attached. Another color-coded approach can be used to identify products that are susceptible to a particular chemical reaction. The approach is to couple the acceptor molecule to an enzyme such as alkaline phosphatase or to a fluorescent, and then add... 

The best-characterized alkaline phosphatase is the enzyme from E. coli, which is a homodimer of 47 kDa subunits. Each subunit contains two Zn ions and one Mg ion, but the metal ions can be readily removed and replaced by other divalent metal ions. The enzyme containing Mg, Co +, or Mn + in the zinc sites has a substantially lower catalytic activity, and the Cd2Mg enzyme has a greatly reduced value of 3, such that the E P species accumulates. 

Phosphorylation of serine residue(s) of the j8-subunit of the carboxyltransferase unit occurs in pea chloroplasts incubated in the light [20]. Alkaline phosphatase treatment reduces ACC activity in parallel to removal of phosphate groups from ACC. This activation by phosphorylation is opposite to the inhibition of animal ACC by phosphorylation but is consistent with the increase in ATP concentration and rates of fatty acid synthesis in chloroplasts in the light and the activation of other plastid enzymes by phosphorylation. These results suggest that the CT subunit reaction is rate determining for overall ACC activity, at least for the multisubunits enzyme of dicots. 

The alkaline phosphatase from E. coli is a zinc-containing metalloenzyme comprising two identical subunits. Different groups have claimed that varying numbers of zinc atoms are present in the dimer, but it has recently been demonstrated that there are four, of which only two appear to... 

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