Alkaline phosphatase phosphoryl enzyme

As well as complexing the substrate to the active site, many enzymes link covalently with the substrate, or a portion of it, to form an additional intermediate. Such intermediates occur in the action of enzymes as diverse as alkaline phosphatase (phosphoryl enzyme), serine and cysteine proteases (acyl enzymes), glycosidases (acylal enzymes) and aldolases. 

Product Acceptors. Many enzyme assays use acceptors, as for instance 2-ethylaminoethanol and other aminated alcohols iihich act as acceptors for the phosphoryl product of the reaction catalyzed by alkaline phosphatase (25) (Fig. 4). Hydroxylamine can act as an acceptor for the hydroxyacetone produced by eno-lase and semicarbazide can act as an acceptor for the pyruvate produced by LD. It is necessary to optimize the concentration of such an acceptor before using it routinely as often what may be a theoretically desirable acceptor is in practice superfluous. 

Alkaline phosphatase catalyzes the dephosphorylation of a mmber of artificial substrates ( ) including 3-glycerophosphate, phenylphosphate, p-nitrophenylphosphate, thymolphthalein phosphate, and phenolphthalein phosphate. In addition, as shown recently for bacterial and human enzymes, alkaline phosphatase simultaneously catalyzes the transphosphorylation of a suitable substance which accepts the phosphoryl radical, thereby preventing the accumulation of phosphate in the reaction mediim (25). 

A more successful strategy for developing sensitive and facile assays to monitor PLCBc activity involves converting the phosphorylated headgroup into a colorimetric agent via a series of enzyme coupled reactions. For example, phosphatidylcholine hydrolysis can be easily monitored in a rapid and sensitive manner by enzymatically converting the phosphorylcholine product into a red dye through the sequential action of alkaline phosphatase, choline oxidase, and peroxidase [33]. This assay, in which 10 nmol of phosphorylcholine can be readily detected, may be executed in a 96-well format and has been utilized in deuterium isotope and solvent viscosity studies [34] and to evaluate inhibitors of PLCBc [33] and site-directed mutants of PLCBc [35,36]. 

Phosphates of pharmaceutical interest are often monoesters (Sect. 9.3), and the enzymes that are able to hydrolyze them include alkaline and acid phosphatases. Alkaline phosphatase (alkaline phosphomonoesterase, EC 3.1.3.1) is a nonspecific esterase of phosphoric monoesters with an optimal pH for catalysis of ca. 8 [140], In the presence of a phosphate acceptor such as 2-aminoethanol, the enzyme also catalyzes a transphosphorylation reaction involving transfer of the phosphoryl group to the alcohol. Alkaline phosphatase is bound extracellularly to membranes and is widely distributed, in particular in the pancreas, liver, bile, placenta, and osteoplasts. Its specific functions in mammals remain poorly understood, but it seems to play an important role in modulation by osteoplasts of bone mineralization. 

F. Hollfelder, D. Herschlag, The Nature of the Transition State for Enzyme-Catalyzed Phosphoryl Transfer. Hydrolysis of O-Aryl Phosphorothioates by Alkaline Phosphatase , Biochemistry 1995, 34, 12255-12264. 

Figure 6.24 Enzyme-labeled fluorescence. ELF-97 is a soluble phosphorylated substrate cleaved by alkaline phosphatase into a highly fluorescent, insoluble product. (Molecular Probes, Inc., Eugene, OR.)...

In subsequent years, much evidence has been adduced to support this mechanism. Alkaline phosphatase and, by analogy, other serine enzymes, are directly phosphorylated on serine serine phosphate is not an artifact (Kennedy and Koshland, 1957). In the presence of nitrophenyl acetate, chymotrypsin is acetylated on serine, and the resulting acetylchymotrypsin has been isolated (Balls and Aldrich, 1955 Balls and Wood, 1956). Similarly, the action of p-nitrophenyl pivalate gave rise to pivaloyl chymotrypsin, which could be crystallized (Balls et al., 1957). Neurath and workers showed that acetylchymotrypsin is hydrolyzed at pH 5.5, but that it is reversibly denatured by 8 M urea the denatured derivative is inert to hydrolysis and even to hydroxylamine, whereas the renatured protein, obtained by... 

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). 

Agren 112) and Engstrom 113) isolated serine phosphate from mammalian alkaline phosphatase that had been incubated with inorganic phosphate in acid pH (<6). Engstrom 114) and Schwartz and Lipmann 35) later obtained similar results with E. coli alkaline phosphatase. They found that a large percentage of the enzyme is phosphorylated, that compounds like glucose 6-phosphate and sodium arsenate inhibit... 

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. 

In order to see if the phosphoryl enzyme is thermodynamically stable as compared to ordinary phosphate esters, Levine et al. (80) carried out kinetic experiments which yielded information concerning the equilibria between Pi and alkaline phosphatase (E) (127). 

In studies with alkaline phosphatase it has been found that the enzymic activity measured by the release of p-nitrophenol from p-nitrophenyl phosphate increases with the concentration of tris buffer much faster than it increases with the ionic strength of other salts such as NaCl and Mg2SO< (4, 50). This behavior of tris was shown by Dayan and Wilson (122, 123) to result from a transphosphorylation reaction, where 0.5 M tris reacts with phosphoryl enzyme to form tris phosphate at the same rate as does 55 M water to form orthophosphate. 

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. 

This feature has been extensively investigated by Engstrom (20, 71, 88, 168, 169 see also Sections I,A and II,B) whose results may be summarized as follows (1) incubation of alkaline phosphatase with 32P-Pi at pH 4—6 and 0°, followed by acid inactivation, leads to the appearance of the label in the enzyme protein (2) after acid hydrolysis the only labeled amino acid found is phosphoserine (3) one mole of Pi is incorporated per mole of enzyme (4) the presence of Zn2+ in the enzyme is essential for phosphorylation (5) bound Pi can be displaced by addition of glucose 6-phosphate to the phosphorylation medium and (6) very little phosphoryl enzyme is formed under alkaline conditions. 

Most of these observations have since been verified. Phosphorylation by substrate has been shown to occur under acid conditions by using a stopped-flow technique (118, 165) as illustrated in Fig. 4. Under alkaline conditions the phosphoryl enzyme cannot normally be observed or isolated because the rate of dephosphorylation exceeds the maximum rate of phosphorylation (170). One interesting aspect is that the pH-rate profiles for phosphorylation and dephosphorylation are quite different, as is the case for E. coli alkaline phosphatase (171). Barman and Gut-freund studied the formation and breakdown of milk phosphoryl phosphatase using a rapid-quenching technique and concluded that dephosphorylation could not be rate limiting for the hydrolysis of p-nitrophenyl phosphate at pH 7 (S3). 

As noted earlier, the binding of Mg2+ to ATP results in activation towards hydrolysis of the terminal phosphoryl group. Hydrolysis and transfer of phosphate may involve transfer of the phosphoryl group to the enzyme, followed by transfer to water or an acceptor molecule. Enzymes such as alkaline phosphatase and fructose-1,6-biphosphatase which require Mg2+ and Zn2+ will be considered in Section 62.1.4. 

Zinc(II) and Co(II) are the only cations found to reactivate apophos-phatase to any appreciable extent (120). The Co(II) enzyme follows the same formal mechanism as the native enzyme, but has a lower specific activity (113, 121). It lacks the phosphotransferase activity (113, 119, 121) observed for the native enzyme, for example in Tris buffers. This was taken to imply that the lower activity of the cobalt enzyme is due to a lower rate of phosphorylation, so that this step becomes rate-limiting also below f>H 7 (113). Stopped-flow experiments by Gottesman etal. (121) show, however, that a very fast burst of -nitrophenol occurs in the cobalt alkaline phosphatase-catalyzed hydrolysis of -nitrophenyl phosphate over a wide pH region. These results strongly suggest that a step subsequent to the phosphorylation is rate-limiting in this metal derivative. 

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. 

The specificity of KD0-8-phosphate phosphatase is shown in Table IV. None of the phosphorylated sugars tested could be dephosphorylated by this enzyme, nor were they inhibitors of the reaction. All of the sugars tested (including KD0-8-phosphate) could be dephosphorylated by alkaline phosphatase. It is important to note, however, that the cells used for the isolation of KD0-8-phosphate phosphatase were grown in the presence of high inorganic phosphate which repressed the synthesis of alkaline phosphatase. It should also be noted that alkaline phosphatase is a well characterized periplasmic enzyme whereas KD0-8-phosphate... 

Digest 1 pg of the pLXSN retroviral plasmid with 1 pi of the restriction enzyme Hpal in 10 pi of 1 x NEBuffer 4 in a 1.5 ml Eppendorf tube at 37°C overnight. This produces a linear 5.9 kb backbone fragment with blunt ends. Add 1 pi of calf intestinal alkaline phosphatase to the tube which removes 5 and 3 phosphoryl groups. This prevents the plasmid from self ligation. 

Tissue concentrations of pyridoxal phosphate are controlled by the balance between phosphorylation and dephosphorylation. The activity of phosphatases acting on pyridoxal phosphate is greater than that of the kinase in most tissues, although this may be an artifact of determining alkaline phosphatase activity at its pH optimum rather than at a more physiological pH, when the two activities are approximately equal. This means that pyridoxal phosphate that is not bound to enzymes is readily dephosphorylated. 

The third example is a phosphoryl transfer enzyme, alkaline phosphatase. The active site of alkaline phosphatase contains two Zn + ions, with a separation of 3.9 A. One zinc center is used to bind the phosphate monoester substrate, the other to activate Ser-102 for nucleophilic attack on the phosphate group of the substrate via an associative mechanism, as shown... 

Another important hydrolytic enzyme of the gut is acid phosphatase Like enterokinasc, it is bound to the enierocyte facing the lumen and is present in the duodenum, jejunum, and ileum. Alkaline phosphatase, a zinc metalloenzyme, also occurs in the gut. Acid phosphatase and alkaline phosphatase catalyze the removal of phosphate groups from a wide variety of compounds in foods, for example, sugar phosphates, triose phosphates, nucleotides such as AMP, ADP, and ATP, pyrophosphate, and phosphorylaled amino adds, A number of sugar and triose phosphates are described in the section on glycolysis in Chapter 4,... 

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. 

Several topoisomerases have been shown to be substrates for protein kinases. Nuclear extracts from a human cell line contain a protein kinase which phosphorylates DNA topoisomerase I from the same cell line (Mills et al., 1982). The type I topoisomerase purified from Novikoff hepatoma cells was found to be a phosphoprotein (Durban et al., 1983). Treatment with alkaline phosphatase dephosphorylates the enzyme and reduces its DNA-relaxing activity. Subsequent treatment with protein kinase restores the activity of the topoisomerase to its original level (Durban et al., 1983). 

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