E. coli, alkaline phosphatase from

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-... 

The X-ray crystal structure of the inorganic phosphate (an inhibitor) complex of alkaline phosphatase from E. coli (9) showed that the active center consists of a Zn2Mg(or Zn) assembly, where the two zinc(II) atoms are 3.94 A apart and bridged by the bidentate phosphate (which suggests a phosphomonoester substrate potentially interacting with two zinc(II), as depicted in Fig. 2), and the Mg (or the third Zn) is linked to one atom of the zinc pair by an aspartate residue at a distance... 

Alkaline phosphatase from E. coli is an enzyme of the 1960 s. Although one brief reference to a phosphatase from E. coli having an alkaline pH maximum was reported in 1933 (1), it was not until the discovery by... 

Early methods of purifying alkaline phosphatase from E. coli involved heat shock (4), disruption of cells with a French press (4, 34, 35), and... 

Purified preparations of alkaline phosphatase from E. coli, judged homogeneous when examined in the analytical ultracentrifuge, contain several isozymes, because several bands which contain enzymic activity are obtained in starch-gel and disc-gel electrophoresis. Although most workers find three bands (38, 39, 41, 43, 69), four (44) and five (70) equally spaced bands have been found. 

Alkaline phosphatases form a well-known class of proteins that perform quite interesting and complicated reactions. As previously reported, Zn enzymes, like carboxypeptidases, thermolysin, and carbonic anhydrases, consist of only one Zn atom per active center. Most of the alkaline phosphatases consist of two 96-kDa subunits, each containing two Zn and one Mg ion. The alkaline phosphatase from E. coli has been crystallized and described in full detail [4], and a mechanism has been proposed. Several enzymes in this category have been mentioned in recent years, some of them also containing different metal ions, such as iron and zinc, as in the purple acid phosphatase [5], It is likely that the detailed structure and mechanism of many more examples of enzymes that remove or add phosphate groups to proteins will become available in the next decade. 

Some insight into the function of the zinc ion in alkaline phosphatase from E. coli has resulted from 35C1 n.m.r. studies.229 The uncertainty which has surrounded the number of zinc ions required for activity of the enzyme has been resolved somewhat by the observation that alkaline phosphatase prepared in the absence of edta requires only 2 moles of Zn2+ per mole of enzyme for full activity. 35C1 line-broadening by n.m.r. shows that on addition of two moles of zinc per mole enzyme, no broadening... 

TSK-DEAE-5PW(weak anion exchange, 5 /i particles) Carboxymethyl dextrans Alkaline phosphatase from E. coli periplasmic space proteins 36... 

Acid and alkaline phosphatases which are believed to pass through a phosphorylated intermediate during the hydrolysis of p-nitrophenolphosphate are inhibited by vanadate )68-69. In the case of alkaline phosphatase from E. Coli, V02+ inhibits better than vanadate(V) (Table 2)68. Typical inhibitor plots are shown in Fig. 10. The reduction of the effectiveness of VOz+ as an inhibitor by about a factor of five in tris buffer probably stems from the complexation of the metal ion by tris. Such complexation is known to occur21. ... 

Alkaline phosphatase from E. coli and secreted alkaline phosphatase (SEAP) from mammalian cells are two examples of secreted reporter proteins (Berger, 1988 Manoil et al., 1990 Yang et al., 1997). Although these reporters are used less frequently, they have the evident advantage that no cell extracts are required. Different substrates allow detection with different detection tools. 

E5. Engstrom, L., Incorporation of inorganic phosphate into alkaline phosphatase from E. coli. Biochim. Biophys. Acta 62, 606-609 (1962). 

The alkaline phosphatase from E. coli is a zinc-containing metalloenzyme comprising two identical subunits. Different groups have claimed that varying numbers of zinc atoms are present in the dimer, but it has recently been demonstrated that there are four, of which only two appear to... 

Alkaline phosphatase from E. coli is another zinc-containing enzyme in which the metal plays an important role (for a review, see ref. 63). Cottam and Ward have recently used C1 n.m.r. spectroscopy to investigate the zinc binding site and have found that the access of Cl to the four metal atoms in the metal-enzyme is severely restricted. They suggest that this result could be explained in two ways. In the first, the protein may provide all of the ligands necessary to completely chelate the zinc ion, thus excluding... 

Halford, S. E., Bennett, N. G., Trentham, D. R. Gutfreund, H. (1969). Substrate induced conformation change in the reaction of alkaline phosphatase from E. coli. Biochemical Journal, 114,243-51. 

The alkaline phosphatase from E. coli is a nonspecific phosphomonoesterase which catalyzes the hydrolysis of 3 - and/or 5 -terminal phosphates from DNA, RNA, and a wide variety of organic phosphomonoesters (Scheme 5.1). The phos-... 

The effect of edta on alkaline phosphatase from E. coli and on the cobalt(ii) and copper(ii) derivatives of the protein has been studied by the measurement of enzyme activity and bye.s.r. spectroscopy. From dialysis experiments on an edta-contaminated apoenzyme, it was found that the edta binds to the metal-free protein. Furthermore, in the complete absence of edta only two Zn + or Co + ions per enzyme molecule were required for full enzyme activity and it is suggested that reports in the literature that more than two metal (ons are necessary (four is the number commonly quoted) may be explained by varying levels of edta contamination in the enzyme and apoenzyme preparations. The ligand edta also affects the e.s.r. spectrum of copper alkaline phosphatase, thus accounting for the two types of signal reported previously and their different behaviour towards phosphate. 

One of the initial routes considered for the preparation of isomers was the enzymatic resolution using 5 -nucleotidase and alkaline phosphatase that would allow access to both enantiomers, racemic compound. The chemically synthesized monophosphate derivative ( )-121 was resolved using 5 -nucleotidase from Crotalus atrox venom, and the resulting mixture was separated by chromatography and purified on silica gel to give (+)-123 with a 90% ee. Hydrolysis of the remaining monophosphate (-)-122 with alkaline phosphatase from E. coli afforded Epivir [155]. 

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