Alkaline phosphatase dimerization

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was also formed, however, a significant amount of enzymatically active protein in which most arginine residues had been replaced by canavanine."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

Chapman and Breslow synthesized zinc(II) complexes of monomer and dimers derived from 1,4,7-triazacyclododecane with phenyl 48 and 4,4 -biphenyl linkers 49 (55). They were examined as catalysts for the hydrolysis of 4-nitrophenyl phosphate (NP2 ) and bis(4-nitrophenyl) phosphate (BNP ) in 20% (v/v) DMSO at 55°C. On the basis of the comparison of the pseudo-first-order rate constants, the dinuclear zinc(II) complexes 48 with 1,3-phenyl and 1,4-phenyl linkers are ca. 5 times more efficient than monomer or 49 in the hydrolysis of NP2, leading to the conclusion that the two zinc(II) ions are simultaneously involved in the hydrolysis, as in the enzyme alkaline phosphatase. For the hydrolysis of BNP, a longer dimer 49 is ca. six times more effective than 1,3-phenyl-linked dimer 48 and monomers. 

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 ... 

By forming spheroplasts from normal cells, Torriani (22) showed that pools of monomer but no alkaline phosphatase (active dimers) exist in the endoplasm and concluded that dimerization occurred outside the endoplasm. Fifteen percent of the enzyme exists as monomers associated with particles in the endoplasm that are larger than ribosomes. 

One possibility is that the concentration of monomers in the endoplasm is too low to produce dimers and that the monomers are concentrated in the periplasmic space by active transport. Evidence against active transport was obtained in episomal transfer of the structural gene from E. coli to S. typhimurium (26). Even though S. typhimurium does not synthesize alkaline phosphatase, the enzyme was produced by the heterogenote and appeared in the periplasmic space. Schlesinger and Olsen (26) argued that it is unlikely that S. typhimurium would have a transport system for alkaline phosphatase monomers because it does not normally make the enzyme. 

Alkaline phosphatase can be reversibly denatured by thiol reduction in the presence of urea (88), a treatment which dissociates the dimer. Proteins purified from alkaline phosphatase-negative mutants that are antigenically related to alkaline phosphatase are readily and reversibly dissociated by acid (65). Normal alkaline phosphatase is more stable but at a lower pH, less than 3.0, it too forms monomers with release of zinc ions. However, chelating agents that remove zinc do not cause... 

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. 

Further studies (38) in vivo and in vitro showed that the new enzymes produced by the cross between E. coli and S. marcescens were a dimer containing one monomer of the E. coli type and one monomer of the S. marcescens type. By crossing E. coli with an S. marcescens mutant that produced very little alkaline phosphatase, it was shown that the alkaline phosphatase isozymes produced gave the same tryptic peptide map as the E. coli wild-type enzyme with the exception of two new peptides. 

There has been some uncertainty concerning the metal content of alkaline phosphatase and the role of zinc in the catalytic process. Early measurements by Plocke et al. (36, 50) showed that there were 2 g-atoms per dimer. The zinc requirement for enzymic activity was demonstrated by the inhibition of the enzyme with metal binding agents in accord with the order of the stability constants of their zinc complexes. It appears that in some cases (EDTA) zinc is removed from the enzyme and in other cases (CN) the ligand adds to the metalloprotein. A zinc-free inactive apoenzyme was formed by dialysis against 1,10-phenanthro-line. Complete activity was restored by zinc only zinc, cobalt, and possibly mercury produce active enzyme. 

Recently, Cottam and Ward (182) found that with the titration of apo-alkaline phosphatase with Zn(II) up to a mole ratio of four Zn(II/ dimer results in no increase in the S5C1 NMR linewidth, .. . while in previous studies of zinc activated biological reactions, a large increase in the chloride linewidth was observed with zinc bound to macromolecules. However, an increase in the chloride linewidth is observed when the pH is decreased below 5.0. This was interpreted as showing that Zn(II) in alkaline phosphatase is not exposed to solvent at pH > 5.0. In an ESR study of Cu(II) binding to alkaline phosphatase, Csopak and Falk (133) reported that two Cu(II) binds to the same specific sites as the two Zn(II), that the ESR spectrum for the one copper enzyme is different from the two copper enzymes, and that phosphate binding causes a shift of the spectral lines. 

The data relevant to the number of active sites of alkaline phosphatase can be divided into two groups One group derived from studies at low substrate concentrations (S < 10-4 M) indicates one active site per dimer, and the other group derived from studies at high substrate concentrations (S > 10 3 M) indicate two sites. 

The dimeric nature of alkaline phosphatase makes it a more complicated system than carbonic anhydrase or carboxypeptidase. The enzyme contains several metal-binding sites. The stoichiometry of zinc binding is not completely settled. There are at least two strongly bound metal ions (109, 111, 114), but the presence of four specific sites has been claimed (115, 116). At alkaline pH, the enzyme tends to bind even more zinc rather strongly, but probably to sites unrelated to catalytic function (109). A critical evaluation of this aspect falls outside the scope of this review, but it appears that some of the apparent discrepancies are due to different experimental methods in measuring metal binding. 

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 are typically dimeric zinc metalloenzymes ranging in size from 80 to 145 kDa. They catalyze a nonspecific phosphomonoesterase reaction of the following type ... 

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... 

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. 

Alkaline phosphatases (AP EC 3.1.3.1) are dimeric, zinc-containing, non-specific phosphomonoesterases which are found in... 

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. 

Recent developments in the structural analyses of enzymes that cleave phosphate esters have revealed a common property for the reaction center (60, 81, 93, 130), namely, more than one metal ion, usually Zn, is involved. The first example of this kind to be identified was E. coli alkaline phosphatase, an isologous dimer of molecular weight 94,058, which contains 4.0 ( 0.3) g atoms of tightly bound Zn " per mole and 1.3 ( 0.2) g atoms of Mg- per mole (130). Recent refinements of the structure of this enzyme (93) containing PO ion show that two phosphate oxygen atoms... 

Bacterial alkaline phosphatase is the gene product of phoA, a member of the pho regu-lon (Table 9.1). When the pho regulon is induced by low external quantities of phosphate, synthesis of this alkaline phosphatase can represent as much as 6 mole% of total protein synthesis, and enzyme activity per cell can increase 1000-fold (Coleman and Gettins, 1983). The enzyme is synthesized as 43,000 Da monomers, which are transported to the periplasmic space and become active only after dimerization. As with many alkaline phosphatases, this enzyme accepts a broad range of substrates, which it hydrolyses at similar rates (Fernley and Walker, 1967 Reid and Wilson, 1971). Substrates are compounds with the general formula... 

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