Serine residues alkaline phosphatase

The active sites of these enzymes can have a nitrogen ligand, usually as histidine (acid phosphatases and some protein phosphatases), a nucleophilic serine residue (alkaline phosphatases), a cysteine residue in which the thiol group can form a covalent species with the phosphate ester (protein phosphatases), or an aspartate-linked phosphate (plasma membrane ion pumps). The inhibitory form of vanadium is usually anionic vanadate V(V), but cationic vanadyl V(IV) has also shown strong inhibition of some types of phosphorylase reactions. Above neutral pH, speciation of vanadyl ions produces anionic V(IV) species capable of inhibition of enzymes in the traditional transition-state analogue manner [5],... 

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 ... 

The amino acid sequence around the serine that is phosphorylated in the presence of inorganic phosphate at low pH can be seen in Table III (55-57). The sequence of Schwartz et al. (55) accounted for 56% of the peptides that contained 32P (20% or more of the peptides were excluded as extreme fractions when the peaks were pooled). The sequence, as far as it is known, is the same for alkaline phosphatase from a mammalian source (58). It is interesting to note, as pointed out by Boyer and others (59-64), that many hydrolytic enzymes with a serine residue at their active site have the same general sequence, i.e., Asp (Glu)-Ser-Ala (Gly). 

With the establishment of the phosphoryl enzyme, the question was whether or not the phosphoryl enzyme was the same as the phospho-protein found by incubating inorganic phosphate with alkaline phosphatase at low pH (35, 114-116, 119, 120). Wilson and Dayan (105) pointed out that the phosphoprotein is thermodynamically very stable It is 105 times more stable than O-phosphorylserine (125) and 0-phosphoryl ethanolamine (105, 126). Alkaline phosphatase, as a true catalyst, must catalyze both the hydrolysis and the formation of phosphate esters. Therefore, if a serine residue existed which was capable of forming a thermodynamically stable phosphate ester, alkaline phosphatase as a nonspecific catalyst would catalyze its formation from both inorganic phosphate and phosphoester substrates. 

More recently, isotopic labeling experiments have assumed a major role in establishing the detailed mechanism of enzymic action. It was shown that alkaline phosphatase possesses transferase activity whereby a phos-phoryl residue is transferred directly from a phosphate ester to an acceptor alcohol (18). Later it was found that the enzyme could be specifically labeled at a serine residue with 32P-Pi (19) and that 32P-phosphoserine could also be isolated after incubation with 32P-glucose 6-phosphate (20), providing strong evidence that a phosphoryl enzyme is an intermediate in the hydrolysis of phosphomonoesters. The metal-ion status of alkaline phosphatase is now reasonably well resolved (21-23). Like E. coli phosphatase it is a zinc metalloenzyme with 2-3 g-atom of Zn2+ per mole of enzyme. The metal is essential for catalytic activity and possibly also for maintenance of native enzyme structure. 

The Escherichia coli alkaline phosphatase (li coli AP) is the most extensively studied phosphatase, and perhaps the most studied two-metal ion catalyst.68,91 95 The AP-catalyzed reaction proceeds via an intermediate in which a serine residue (Ser-102 in E. coli AP) is phosphorylated. Thus, the stereochemical outcome of the overall reaction is retention. The hydrolysis of the intermediate by water to produce inorganic phosphate competes with phosphoryl transfer to other acceptors such as alcohols or nucleophilic buffers if such are present in solution. The rate-limiting step... 

Osteoblasts secrete osteoid, a matrix rich in type I collagen fibers and vesicles. Precipitation of calcium phosphate is inhibited by a high concentration of pyrophosphate in stromal interstitial fluids, and a high concentration also of albumin and citrate in blood plasma. Pyrophosphate is derived from (1) transport out of the cytosol, and (2) synthesis from nucleoside triphosphates in the stromal interstitial fluid that permeates the osteoid matrix. Precipitation occurs only when calcium and phosphate ions are taken up into vesicles whose inner membrane is composed of phosphatidylserine. The high concentration of calcium and phosphate ions in the vesicle is mediated by annexin and type HI Pi Na-dependent transporters. This overwhelms the pyrophosphate and nucleation occurs. As the precipitate grows and ruptures the membrane, tissue-nonspecific alkaline phosphatase is activated to remove pyrophosphate from the osteoid matrix fluid so that calcium phosphate precipitates around phosphorylated serine residues within the collagen fibers. 

M20. Milstein, C., The amino acid sequence around the reactive serine residue in alkaline phosphatase from Escherichia coli. Biochem. J. 92, 410-421 (1964). 

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. 

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