Alkaline phosphatase transphosphorylation

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). 

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. 

Since the equilibrium lies well to the right it is customary to say that alkaline phosphatase hydrolyzes phosphate esters, but some related compounds are also hydrolyzed (Table VI) 3, 4, 28, SO, 94-100). The enzyme also catalyzes transphosphorylation reactions in which a different alcohol substitutes for H20 as a phosphate acceptor. Compounds that are hydrolyzed have the general structure,... 

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. 

J.R. Knowles and coworkers used chiral [l60, l70, lsO]phosphate esters to show that the transphosphorylation catalyzed by E. coli alkaline phosphatase proceeds with overall retention of configuration [52]. They synthesized phenyl-[160, l70, lsO]phosphate by the procedure in Fig. 19 and used it as the phosphoryl-donor substrate with 1,2-propanediol as acceptor according to Equation 12. They determined the configuration of the 1,2-propanediol-1-[16O, l70, lsO]phosphate by the procedure described in Figs. 21 and 22 and found it to be the same as that of the phenyl-[l60, l70, lsO]phosphate they used as the phosphate donor. 

M7. Meyerhof, O., and Green, H., Synthetic action of phosphatase. II. Transphosphorylation by alkaline phosphatase in the absence of nucleotides. J. Biol. Chem. 183, 377-390 (1950). 

WIO. Wilson, I. B., Dayan, J., and Cyr, K., Some properties of alkaline phosphatase from Escherichia coli. Transphosphorylation. J. Biol. Chem. 239, 4182-4185 (1964). 

Fig. 8 Catalytic mechanism of alkaline phosphatase reaction [44]. The initial alkaline phosphatase (E)-cataiyzed reaction consists of a substrate (DO-Pi) binding step, phosphate-moiety transfer to Ser-93 (in the TNAP sequence of its active site), and product alcohol (DOH) release. In the second part of the reaction, phosphate is released through hydrolysis of the covalent intermediate (E-Pi) and non-covaient compiex (E Pi) of inorganic phosphate in the active site. In the presence of alcohol molecules (AOH), phosphate is aiso reieased via a transphosphorylation reaction...

Alkaline phosphatase (AP orthophosphoric monoester phospho-hydrolase, EC 3.1.3.1) isozymes are present in a wide range of species from bacteria to man and are capable of dephosphorylation and transphosphorylation of a broad spectrum of substrates in vitro [1]. Their broad snbstrate specificity and localization on the outside leaf of the cytoplasmic membrane suggests potential involvement in numerons extracellular processes. In humans, four isozymes of APs have been identified. One of them, tissue-nonspecific alkaline phosphatase (TNAP), is ubiquitously expressed, demonstrating especially high level of expression in bone, liver and kidney tissues. Three other isozymes demonstrate tissue-specific... 

Again, the configuration is inverted. Nucleoside diphosphate kinase catalyzes the same transfer, but to a nucleoside diphosphate rather than to AMP, and with retention of configuration rather than with inversion (70). The mechanism of action of adenylate kinase involves a single displacement at P and that of nucleoside diphosphate kinase involves a double displacement at P via an intermediate phosphoenzyme. Although alkaline phosphatase is not classified as a phosphotransferase, it catalyzes transphosphorylation via the same phosphoenzyme that is the intermediate in the phosphatase reaction. This enzyme catalyzes reaction... 

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. 

In the presence of alcohols, alkaline phosphatase displays transphosphorylation activity, i.e., hydrolysis of the starting ester and esterification of the phosphate group with a different alcohol. This ability is easily understood if one keeps in mind that the reaction depicted above is reversible, and that a different alcohol may be involved in the formation of the ester bond. Most group-transfer reactions catalyzed by metalloenzymes are likely to proceed through the same elementary steps proposed for hydrolytic reactions. 

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