Alkaline phosphatase mechanism

Alkaline phosphatase, 2,774 mechanism, 6,612 metal substitution, 6,611 structure, 6,611 zinc, 5.1006 6,610 Alkanes... 

Figure 12.12 Principal steps in the mechanism of alkaline phosphatase. (Reprinted with permission from Parkin, 2004. Copyright (2004) American Chemical Society.)...

Finally we should briefly mention the purple acid phosphatases, which, unlike the alkaline phosphatases, are able to hydrolyse phosphate esters at acid pH values. Their purple colour is associated with a Tyr to Fe(III) charge transfer band. The mammalian purple acid phosphatase is a dinuclear Fe(II)-Fe(III) enzyme, whereas the dinuclear site in kidney bean purple acid phosphatase (Figure 12.13) has a Zn(II), Fe(III) centre with bridging hydroxide and Asp ligands. It is postulated that the iron centre has a terminal hydroxide ligand, whereas the zinc has an aqua ligand. We do not discuss the mechanism here, but it must be different from the alkaline phosphatase because the reaction proceeds with inversion of configuration at phosphorus. 

Figure 2 illustrates the reaction mechanisms of acridinium ester label probes and alkaline phosphatase probes using dioxetane chemiluminescent detection. Table 2 summarizes approaches for labeling DNA. 

The phosphorescence lifetimes of various proteins at room temperature are given in Table 3.1. Some variability in the lifetimes reported from lab to lab is evident, possibly due to different enzyme preparation, removal of oxygen (see below), or other conditions. Nevertheless, when measured under the same conditions, it is apparent that the tryptophan lifetimes vary dramatically from protein to protein. Alkaline phosphatase exhibits the longest lifetime from a protein in solution with a lifetime of 1.5—1.7 s at 22°C, approaching the lifetime of 5.5 s at 77 K. The lifetime of free indole in solution is 15—30 /is at 22°C.(38 39) Therefore, in the absence of other quenching mechanisms, the lower limit for the phosphorescence lifetime of a fully exposed tryptophan moiety in a protein should be about 20 /is. 

J. E. Coleman, P. Gettins, Alkaline Phosphatase, Solution Structure and Mechanism , Adv. Enzymol. Relat. Areas Mol. Biol. 1983, 55, 381-452. 

Fig. 9.7. a) Mechanism of activation of (phenytoin-3-yl)methyl phosphate (9.25) to release phenytoin. Phosphoric acid ester hydrolysis is mediated by alkaline phosphatase, b) (Phospho-ryloxy)methyl prodrugs of tertiary amines, whose activation occurs by the same two-step mechanism shown in a [79]. 

At-MNDP 31-33, 36, 108), whose mechanism of intracellular localization is related to the presence of oncogenically expressed tumor-membrane alkaline phosphatase isoenzymes 42, 108), has been demonstrated strikingly effective in an animal tumor model 33, 34, 38, 39). It has also served as a concomitant analytical probe for identifying the intracellular locus of radiotherapeutic action of this class of drug by a-particle track autoradiography 33,106-109). Phase I and II human therapeutic trials are shortly envisaged 33, 34). 

Liver injury is clinically defined as an increase of serum alanine amino transferase (ALT) levels of more than three times the upper limit of normal and a total bilirubin level of more than twice the upper limit of normal [4]. The clinical patterns of liver injury can be characterized as hepatocellular (with a predominant initial elevation of ALT), cholestatic (with an initial elevation of alkaline phosphatase) or mixed. The mechanisms of drug-induced hepatotoxicity include excessive generation of reactive metabolites, mitochondrial dysfunction, oxidative stress and inhibition of bile salt efflux protein [5]. Better understandings of these mechanisms in the past decades led to the development of assays and models suitable for studying such toxic mechanisms and for selecting better leads in the drug discovery stage. 

Table 2 Effects of alkaline phosphatase concentration on the gelation time and mechanical properties of the peptide hydrogels of Emoc-Y-OH (entries 1-4) and...

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... 

FIGURE 19. Reaction mechanism of alkaline phosphatase. For clarity, the complete active site is shown only in the first strucmre. Adapted with permission from Reference 10. Copyright (2005) ACS... 

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). 

Alkaline phosphatase (AP) is a (Znn)2-containing phosphomonoester-ase that hydrolyzes phosphomonoesters (RO—POf-) at alkaline pH (7). Ser102 under the influence of one of the zinc(II) ions at the active center 1 (Fig. 2) is directly involved in phosphate hydrolysis (8). On the basis of X-ray structure and NMR studies (9), the mechanism now accepted is that the phosphate substrate, initially recognized by cooperative... 

The two-step mechanism of phosphate ester hydrolysis by the (Znn)2-containing alkaline phosphatase (AP) (7) is thus somewhat mimicked by 24. The phosphoryl intermediate 25 is generated by nucleophilic attack of the alkoxide moiety in 24b at BNP" and is hydrolyzed by the intramolecular Zn11—OH" species in 25b. Thus, the attack at the BNP... 

The two-metal mechanisms have been known for most phosphotransferases e.g., alkaline phosphatase (7), inositol monophosphatase... 

The mechanism by which the more recently used WR-2721 (Fig. 8) reduces nephrotoxicity is not very well understood. WR-2721 protects against nephrotoxicity when administered just prior to cis-Pt (144,145) and it is known that WR-2721 is preferentially taken up by normal cells and not by tumor cells (146). Recently, it was concluded that the uncharged form of the dephosphorylated WR-2721 (known as WE-1065) is the actual species taken up by both normal and tumor cells (147). It has been proposed that the conversion of WR-2721 to WR-1065 is slower in tumors, compared with normal tissues (147), possibly because tumors generally have lower levels of alkaline phosphatase (148). Furthermore, it has been proposed (147) that once formed, WR-1065 will have a decreased uptake rate in tumors, probably as a consequence of their lower pH (149) as compared with normal tissues i.e., the neutral form of WR-1065 will only constitute 0.1% of the total drug present at pH 7 and 1% of the total at pH 8. The reactive WR-1065 is likely to bind directly to cis-Pt, thereby preventing side reactions of cis-Pt. 

Although inversion was not observed with the E. colt alkaline phosphatase, it has been observed for ribonucleases and many other hydrolytic enzymes and for most kinases transferring phospho groups from ATP. The difference lies in the existence of a phospho-enzyme intermediate in the action of alkaline phosphatase (see Eq. 12-38). Each of the two phosphotransferase steps in the phosphatase action apparently occurs with inversion. The simplest interpretation of all the experimental results is that phosphotransferases usually act by in-line -like mechanisms which may involve metaphosphate-ion-like transition states that are constrained to react with an incoming nucleophile to give inversion. An adjacent attack with pseudorotation would probably retain the original configuration and is therefore excluded. 

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 ... 

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