Alkaline phosphatase phosphate hydrolysis with

Deming and Pardue studied the kinetics for the hydrolysis of p-nitrophenyl phosphate by the enzyme alkaline phosphatase. The progress of the reaction was monitored by measuring the absorbance due to p-nitrophenol, which is one of the products of the reaction. A plot of the rate of the reaction (with units of pmol mL s ) versus the volume, V, (in milliliters) of a serum calibration standard containing the enzyme yielded a straight line with the following equation... 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

A determination of the pH dependence of the lanthanum hydroxide gel-promoted hydrolysis of /3-glyceryl phosphate revealed that the two maxima exist in the pH-rate profile, one at pH 8.6 which presumably involves the species La (OH)+2 and another (smaller) maximum at pH 10.4 which involves the species La (OH) 2+ (4). Presumably the same kind of catalytic mechanism is operative in both cases. These reactions may serve as models for the metal ion-promoted alkaline phosphatases which have been shown to proceed with P—O cleavage (and with no oxygen exchange). 

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. 

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. 

Tris and glycerol are not acceptors with Co(II) alkaline phosphatase over the pH range 4.5-11, yet the Co (II) enzyme, like the Zn(II) enzyme, catalyzes the hydrolysis of different phosphate esters at the same rate (91, 94, 96). Also, the Km values of various substrates show little... 

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. 

An important inhibitor of alkaline phosphatase is Pi, normally a very effective competitor with an affinity comparable to that for good substrates (107, 117). In consequence the kinetics of hydrolysis are often approximately first order whatever the substrate concentration, and for this reason initial rate measurements should be limited to 10% hydrolysis. With poor substrates, e.g., phosphocreatine (113) or o-carboxyphenyl phosphate (176), additional care is required. Arsenate is an even more powerful competitive inhibitor (101a, 118) and can prevent the incorporation of Pi by intestinal phosphatase at pH 5 (169). Phosphonates have been reported as weak inhibitors of intestinal phosphatase (120), and one has been used recently as a chromophoric probe for E. coli phosphatase (177). 

These results are common with proteases and also for the hydrolysis of phosphate esters catalysed by alkaline phosphatase in the latter case, there is a pitfall in that the constant value for kcat for phosphate esters is due to a rate-limiting conformational change in... 

Artificial enzymes with metal ions can also hydrolyze phosphate esters (alkaline phosphatase is such a natural zinc enzyme). We examined the hydrolysis of p-nitro-phenyfdiphenylphosphate (29) by zinc complex 30, and also saw that in a micelle the related complex 31 was an even more effective catalyst [118]. Again the most likely mechanism is the bifunctional Zn-OH acting as both a Lewis acid and a hydroxide nucleophile, as in many zinc enzymes. By attaching the zinc complex 30 to one or two cyclodextrins, we saw even better catalysis with these full enzyme mimics [119]. A catalyst based on 25 - in which a bound La3+ cooperates with H202, not water - accelerates the cleavage of bis-p-nitrophenyl phosphate by over 108-fold relative to uncatalyzed hydrolysis [120]. This is an enormous acceleration. 

Figure 9.133 HPLC separation of (/4) guanosine nucleotides and guanosine, injected amount 5 to 10 nmol each in 5 /xL, and (B) neopterin phosphates. The mixture of neopterin phosphates injected was produced by partial hydrolysis of 16 pmol of N IP with alkaline phosphatase and addition of 2, 3 -cNMP. (From Blau and Nieder-wieser, 1983.)...

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