Alkaline phosphatase , zinc enzyme

Metabolic Functions. Zinc is essential for the function of many enzymes, either in the active site, ie, as a nondialyzable component, of numerous metahoenzymes or as a dialyzable activator in various other enzyme systems (91,92). WeU-characterized zinc metahoenzymes are the carboxypeptidases A and B, thermolysin, neutral protease, leucine amino peptidase, carbonic anhydrase, alkaline phosphatase, aldolase (yeast), alcohol... 

One of the most important metals with regard to its role in enzyme chemistry is zinc. There are several significant enzymes that contain the metal, among which are carboxypeptidase A and B, alkaline phosphatase, alcohol dehydrogenase, aldolase, and carbonic anhydrase. Although most of these enzymes are involved in catalyzing biochemical reactions, carbonic anhydrase is involved in a process that is inorganic in nature. That reaction can be shown as... 

While having three metal ions in an enzyme active site is uncommon, it is not unique to PLCBc. The well-known alkaline phosphatase from E. coli (APase) contains two zinc ions and a magnesium ion [67], whereas the a-toxin from Clostridiumperfringens [68]. and the PI nuclease from Penicillium citrinum [69] each contain three zinc ions. Indeed, the zinc ions and coordinating ligands of PI nuclease bear an uncanny resemblance to those of PLCBc as the only differ-... 

It was clear for some time that a number of zinc enzymes required two or more metal ions for full activity, but in the absence of X-ray structural data the location of these metal centres with regard to one another was often uncertain. When the first 3-D structures began to appear, it became clear that the metals were in close proximity. A particular feature of many of these enzymes was the presence of a bridging ligand between two of the metal sites, usually an Asp residue of the protein, which is occasionally replaced by a water molecule. While some of the sites contain only Zn ions, several contain Zn in combination with Cu (in cytosolic superoxide dismutases) Fe (in purple acid phosphatases) or Mg (in alkaline phosphatase and the aminopeptidase of lens). 

Several zinc enzymes that catalyse the hydrolysis of phosphoesters have catalytic sites, which contain three metal ions in close proximity (3-7 A from each other). These include (Figure 12.11) alkaline phosphatase, phospholipase C and nuclease PI. In phospholipase C and nuclease PI, which hydrolyse phosphatidylcholine and single-stranded RNA (or DNA), respectively, all three metal ions are Zn2+. However, the third Zn2+ ion is not directly associated with the dizinc unit. In phospholipase C, the Zn-Zn distance in the dizinc centre is 3.3 A, whereas the third Zn is 4.7 and 6.0 A from the other two Zn2+ ions. All three Zn2+ ions are penta-coordinate. Alkaline phosphatase, which is a non-specific phos-phomonoesterase, shows structural similarity to phospholipase C and PI nuclease however,... 

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. 

Currently, only a handful of examples of unique protein carboxylate-zinc interactions are available in the Brookhaven Protein Data Bank. Each of these entries, however, displays syn coordination stereochemistry, and two are bidentate (Christianson and Alexander, 1989) (Fig. 5). Other protein structures have been reported with iyw-oriented car-boxylate-zinc interactions, but full coordinate sets are not yet available [e.g., DNA polymerase (Ollis etal., 1985) and alkaline phosphatase (Kim and Wyckoff, 1989)]. A survey of all protein-metal ion interactions reveals that jyw-carboxylate—metal ion stereochemistry is preferred (Chakrabarti, 1990a). It is been suggested that potent zinc enzyme inhibition arises from syn-oriented interactions between inhibitor carboxylates and active-site zinc ions (Christianson and Lipscomb, 1988a see also Monzingo and Matthews, 1984), and the structures of such interactions may sample the reaction coordinate for enzymatic catalysis in certain systems (Christianson and Lipscomb, 1987). 

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. 

Zinc is essential for the function of more than 100 enzymes (e.g., thymidine kinase, carbonic anhydrase, lactic dehydrogenase, alkaline phosphatase) involved in a variety of metabolic activities in the body,... 

The most important chemical function of Zn2+ in enzymes is probably that of a Lewis acid providing a concentrated center of positive charge at a nucleophilic site on the substrate/ This role for Zn2+ is discussed for carboxypeptidases (Fig.12-16) and thermolysin, alkaline phosphatase (Fig. 12-23),h RNA polymerases, DNA polymerases, carbonic anhydrase (Fig. 13-1),1 class II aldolases (Fig. 13-7), some alcohol dehydrogenases (Fig. 15-5), and superoxide dismutases (Fig.16-22). Zinc ions in enzymes can often be replaced by Mn2+, Co2+, and other ions with substantial retention of catalytic activity/ ... 

We shall now briefly outline some of the features of the zinc metalloenzymes which have attracted most research effort several reviews are available, these are indicated under the particular enzyme, and for more detailed information the reader is referred to these. Attention is focussed here, albeit briefly, on carbonic anhydrases,1241,1262,1268 carboxypeptidases, leucine amino peptidase,1241,1262 alkaline phosphatases and the RNA and DNA polymerases.1241,1262,1462 Finally, we examine alcohol dehydrogenases in rather more detail to illustrate the use of the many elegant techniques now available. These enzymes have also attracted much effort from modellers of the enzymic reaction and such studies, which reveal much interesting coordination chemistry and often new catalytic properties in their own right—and often little about the enzyme system itself (except to indicate possibilities), will be mentioned in the next section of this chapter. 

These enzymes, which mainly catalyze hydrolytic reactions, have the zinc ions at their active sites. However, Zn ions also appear necessary in some cases for stabilization of the protein structure, e.g. in Cu/Zn SOD, insulin, liver alcohol dehydrogenase and alkaline phosphatases. 

Photooxidation of alkaline phosphatase in the presence of methylene blue and Rose Bengal causes loss of activity for both native and apo-enzyme. In the case of the native enzyme, zinc protects 2 to 3 of the 16 histidine residues. The rate of oxidation of tryptophan is not affected by zinc, and there was no loss of tyrosine. Also, photooxidation of the apoenzyme diminishes zinc binding. It would appear that histidine residues play a role in binding the two zinc ions necessary for enzymic activity (91). 

There has been some uncertainty concerning the metal content of alkaline phosphatase and the role of zinc in the catalytic process. Early measurements by Plocke et al. (36, 50) showed that there were 2 g-atoms per dimer. The zinc requirement for enzymic activity was demonstrated by the inhibition of the enzyme with metal binding agents in accord with the order of the stability constants of their zinc complexes. It appears that in some cases (EDTA) zinc is removed from the enzyme and in other cases (CN) the ligand adds to the metalloprotein. A zinc-free inactive apoenzyme was formed by dialysis against 1,10-phenanthro-line. Complete activity was restored by zinc only zinc, cobalt, and possibly mercury produce active enzyme. 

Simpson and Vallee (51) found that when alkaline phosphatase is exposed to 8-hydroxyquinoline-5-sulfonic acid, two zincs are rapidly removed and the enzyme is inactivated to within 10%. The two remaining zincs are removed more slowly, presumably with the loss of the remaining activity. 

Recently, Cottam and Ward (182) found that with the titration of apo-alkaline phosphatase with Zn(II) up to a mole ratio of four Zn(II/ dimer results in no increase in the S5C1 NMR linewidth, .. . while in previous studies of zinc activated biological reactions, a large increase in the chloride linewidth was observed with zinc bound to macromolecules. However, an increase in the chloride linewidth is observed when the pH is decreased below 5.0. This was interpreted as showing that Zn(II) in alkaline phosphatase is not exposed to solvent at pH > 5.0. In an ESR study of Cu(II) binding to alkaline phosphatase, Csopak and Falk (133) reported that two Cu(II) binds to the same specific sites as the two Zn(II), that the ESR spectrum for the one copper enzyme is different from the two copper enzymes, and that phosphate binding causes a shift of the spectral lines. 

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. 

Alkaline phosphatases form a widespread group of relatively unspecific enzymes catalyzing the hydrolysis of many orthophosphate monoesters. Their pH optima are generally at pH 8 or above. Several alkaline phosphatases have been shown to contain zinc (3). 

The dimeric nature of alkaline phosphatase makes it a more complicated system than carbonic anhydrase or carboxypeptidase. The enzyme contains several metal-binding sites. The stoichiometry of zinc binding is not completely settled. There are at least two strongly bound metal ions (109, 111, 114), but the presence of four specific sites has been claimed (115, 116). At alkaline pH, the enzyme tends to bind even more zinc rather strongly, but probably to sites unrelated to catalytic function (109). A critical evaluation of this aspect falls outside the scope of this review, but it appears that some of the apparent discrepancies are due to different experimental methods in measuring metal binding. 

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. 

On the other hand, alkaline phosphatase may have two equivalent active sites which are coupled so that, normally, only one can operate at a time. This seems an attractive alternative for an enzyme consisting of two identical subunits. In a preliminary paper, Lazdunski et al. (125) report the covalent incorporation of two phosphates into the zinc enzyme as well as the cobalt enzyme, at >H<4. At these low pH values, the free enzyme generally loses its metal ions and dissociates into monomeres (109). However, if these results are corroborated after the performance of proper controls, and if both phosphates are linked to specific amino acid residues in the enzyme, conditions may have been found for the uncoupling of active sites in alkaline phosphatase. 

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. 

It is likely that at low zinc concentrations there is impairment of the activity of vital zinc metalloenzymes such as lactic dehydrogenase, alkaline phosphatase, carbonic anhydrase, carboxypeptidase, and of enzymes in which zinc acts as a cofactor. Injection experiments showed that radioactive 65Zn preferentially concentrated in healing tissues27. ... 

We have already seen a number of models for the zinc(II) containing enzymes such as carbonic anhydrase in Section 11.3.2. Zinc is an essential component in biochemistry, and forms part of the active site of more then 100 enzymes, of which hydrolases (such as alkaline phosphatase and carboxypeptidase A), transferases (e.g. DNA and RNA polymerase), oxidoreductases (e.g. alcohol dehydrogenase and superoxide dismutase) and lysases (carbonic anhydrase) are the most common. In addition, the non-enzyme zinc finger proteins have an important regulatory function. In many of these systems, the non-redox-active Zn2+ ion is present as a Fewis acidic centre at which substrates are coordinated, polarised and hence activated. Other roles of zinc include acting as a template and playing a structural or regulatory role. 

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