Active site alkaline phosphatase

Figure 17-7 Two alternative mechanisms utilized by phosphatases to carry out hydrolysis of phosphate esters. The phosphoenzyme intermediate mechanism utilizes an amino acid (represented as -X] as a nucleophile to attack the phosphate ester, transferring the phosphoryi group and producing a short-lived phosphoenzyme intermediate. In the second step, water serves as the nucleophile, hydrolyzing the phosphoenyzme intermediate and regenerating the enzyme. This mechanism is used by the tyrosine phosphatases (nucleophile = cysteine) and E. coli alkaline phosphatase (active site nucleophile = Ser 102). The metallophosphatases do not proceed by formation of a phosphoenzyme intermediate but rather carry out hydrolysis by direct transfer of the phosphoryi group to a metal-coordinated water molecule.

Fig. 3. Hydrolysis of 4-methylumbelliferyl phosphate by calf intestinal alkaline phosphatase. Activities are recorded as turnovers per site per second at 20° and 1 = 0.02, using tris-acetic acid (< pH 8) or ammediol-HCl (> pH 8) buffers.

Using the Bodansky (B18, 52) procedure with 8-glycerophosphate as substrate, Woodard (W8) was unable to obtain such elevations. She determined the serum acid phosphatase activities in 83 females and 342 males, or a total of 425 patients with miscellaneous diseases. Of these, 61 had various types of infectious or metabolic disorders, including 11 cases of inflammatory disease of bone and 12 cases of hepatic cirrhosis. The remainder had some type of neoplastic disease and about one-third had metastases to bone from cancer of various primary sites. There were 15 cases of osteogenic sarcoma and 32 cases of osteitis deformans. All these cases, whether their serum alkaline phosphatase activities were elevated or not, had serum acid phosphatase values that were essentially within the normal range, 0.06-0.89 Bodansky unit for females and 0.11-0.88 unit for males. In contrast to the Gutman method (GIO, G14), there-... 

The safety and activity of subcutaneous GM-CSF (300 micrograms/day for 1 week and 150 micrograms twice weekly for 11 weeks) has been compared with no treatment in 244 leukopenic HIV-infected patients (62). Adverse effects were reported in most of the patients treated with GM-CSF and consisted of flu-like symptoms (98%), bone pain (42%), and injection site reactions (85%). There was a two-fold increase in serum transaminase and alkaline phosphatase activities in 5.7% of patients. There was a moderate, but not significant, increase in HIV p24 antigen concentration. The few relevant clinical trials have provided no convincing evidence that GM-CSF enhances HIV replication or accelerates HIV-associated diseases (for example infections or neoplasms) in patients with AIDS (63). Only one patient with AIDS and ultrasonographic confirmation of enhanced Kaposi s sarcoma lesions temporally related to GM-CSF used for interferon- and zidovudine-related severe neutropenia has been reported (SEDA-19, 343). 

Sites of alkaline phosphatase activity are frequently in endothelial cells of blood capillaries, mucous glandular cells (F3), microvilli of intestine (C6, CIO, D2, H21, P8, W7), bile canaliculi (D21, F27, W2), and placenta (W3), as well as in the brush border of the lumenal surface of epithelial cells of the proximal convoluted renal tubules (M22, Wl). The location of L-phenylalanine-sensitive alkaline phosphatase in human intestine and placenta is illustrated in Fig. 30. Electron micrographs (Fig. 31) show the details of the alkaline phosphatase, and illustrate the... 

Serum alkaline phosphatase estimations are less reliable in the detection of skeletal metastases than are bone scintigrams (03). More than 30% of patients with positive bone scans have normal serum alkaline phosphatase activities (C35). Patients with skeletal metastases from prostatic carcinoma have higher mean serum alkaline phosphatase values than do patients with skeletal metastases derived from other sites (S17), particularly when hypocalcemia is also present (R5). It has been suggested (L21) that prostatic metastases cause oncogenic osteomalacia but serum alkaline phosphatase measurements do not distinguish patients with this complication from those without (L21). The use of serum alkaline phosphatase measurements in carcinoma of the breast is discussed in Section 10.1. 

Hernandez, I., Christmas, M., Yelloly, j.M. and Whitton, B.A. (1997) Factors affecting surface alkaline phosphatase activity in the brown alga Fucus spiralis L. studies at a North Sea intertidal site (Tyne Sands, Scotland), youma/ of Phycology 33, 559-575. 

Plate 20 An example of a fluorescent molecular probe, Endogenous alkaline phosphatase activity in the zebraflsh brain was localized with an endogenous phosphatase detection kit. Enzymatic cleavage of the phosphatase substrate yields a bright yellow-green fluorescent precipitate at the site of enzyme activity. See Fluorescent Molecular Probes. Reproduced with permission from Greg Cox, Molecular Probes, Inc. 

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... 

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]. 

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). 

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). 

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