Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The second-order rate constant, kcat/km, for catalysis of the hydrolysis of 4-nitrophenyl phosphate by alkaline phosphatase decreases with increasing viscosity in the presence of sucrose or arabinose, with a slope of delta[kcat/Km)0/(kcat/Km)]/delta(eta/eta 0) = 1.4 at pH 8.0, 25 degrees C. This is consistent with rate-limiting diffusional encounter of the substrate with active enzyme and indicates that alkaline phosphatase is a "perfect enzyme". However, the reported second-order rate constants of kcat/Km = 6.6 x 10(6) to 4.6 x 10(7) M-1 s-1 are smaller than the diffusional limit; this shows that only approximately 0.1-1% of the diffusional encounters are productive. The first-order rate constant, kcat, for rate-limiting hydrolysis of the phosphoenzyme intermediate at pH = 6 with saturating substrate concentration is independent of viscosity in aqueous sucrose solutions. This shows that sucrose does not destabilize the transition state for phosphoenzyme hydrolysis. However, at pH 8.0 product dissociation is rate limiting and kcat decreases with increasing viscosity in the presence of sucrose, with slopes of delta(k0/kobsd)/delta(eta/eta 0) = 1.2 in 0.04 M Mops buffer, 1.0 in 0.1 M Tris, and 1.2 in 0.67 M Tris buffer. This is consistent with rate-limiting diffusional separation of inorganic phosphate and of Tris phosphate from the enzyme. In contrast, glycerol causes a large decrease in kcat/Km at pH 8.0 and also decreases kcat at pH 6. This shows that glycerol decreases the rate by a solvent effect on the catalytic activity of the enzyme, as well as by increasing the viscosity.
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PMID:Alkaline phosphatase is an almost perfect enzyme. 806 74

Alkaline phosphatase was used as a model in studies to assess the effects of lyophilization on biological activity and molecular integrity in the presence or absence of added carbohydrate. The stability of the activity of alkaline phosphatase, lyophilized in Tris buffer alone or in the presence of the carbohydrates mannitol, lactose or trehalose was examined. Enzyme activity in formulations with Tris buffer alone or with mannitol was considerably reduced by freeze-drying and further storage at elevated temperatures; freeze-drying with mannitol failed to maintain activity at a temperature of 37 degrees C over 21 days, whilst the loss of activity was more gradual when freeze-dried in buffer alone and stored at higher temperatures. Lactose and trehalose maintained the alkaline phosphatase activity after freeze-drying and, furthermore, preparations containing trehalose retained activity even when the material was subjected to temperatures of up to 45 degrees C for up to 84 days. At 56 degrees C the alkaline phosphatase activity did not show a significant drop until 14 days with the lactose formulation or until 21 days with trehalose. After 84 days at 56 degrees C, 30% of the activity still remained in the formulation containing trehalose. In addition to the changes in the enzyme activity, FPLC chromatographic traces and SDS-PAGE gels demonstrated compositional differences between each formulation after storage.
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PMID:The effect of carbohydrate additives in the freeze-drying of alkaline phosphatase. 809 38

In the X-ray structure of Escherichia coli alkaline phosphatase at 2.0-A resolution, His-372 was found only 3.8 A away from the zinc and forms a hydrogen-bonding interaction with Asp-327, a bidentate ligand of the zinc at the M1 site. However, His-372 does not directly interact with the zinc atom at the M1 site. In order to investigate the role of the side chain of His-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase, site-directed mutagenesis was used to convert His-372 to alanine. The fact that the His-372-->Ala enzyme has similar zinc binding affinity as the wild-type enzyme indicates that His-372 is not involved in zinc binding at the M1 site. However, the altered kinetic behavior of the mutant enzyme compared to the wild-type enzyme suggests that the imidazole ring of His-372 plays an indirect role in the catalytic mechanism of the enzyme. The hydrolysis activity of the His-372-->Ala enzyme at pH 8.0 is 10-fold lower than that of the wild-type enzyme. In the presence of a phosphate acceptor at pH 8.0, the mutant enzyme is approximately 80% as active as the wild-type enzyme. Therefore, the His-372-->Ala mutation selectively enhances the transphosphorylation activity of the enzyme. The His-372-->Ala enzyme also exhibits 4- and 30-fold decreases in Km as compared to the wild-type enzyme in 0.1 M MOPS buffer and 1.0 M Tris, buffer at pH 8.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Probing the role of histidine-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase by site-specific mutagenesis. 811 85

Major histocompatibility (MHC) class II antigens are heterodimeric cell surface glycoproteins consisting of an alpha and a beta chain. Although one-dimensional SDS-polyacrylamide gel electrophoresis analysis of purified MHC class II antigens shows a single diffuse band for each chain, multiple spots of identical molecular size were observed for each chain when analyzed by two-dimensional electrophoresis. The basis of this heterogeneity has not been clearly defined and has been predicted partially to be due to glycosylation and/or phosphorylation of the mature protein. To investigate the role of the three N-linked oligosaccharides of the alpha and beta chains in determining the isoelectric point of each chain, affinity-purified MHC class II antigens from human and rat sources were deglycosylated using asparagine amidase. The complete enzymatic removal of all three N-linked oligosaccharides was confirmed by SDS-polyacrylamide gel electrophoresis as well as by four different lectin-linked Western blot analyses. Two-dimensional gel analysis of the deglycosylated molecules shows no significant difference from the fully glycosylated chains. We have expressed truncated forms of the HLA DR2 chains which lack the transmembrane and cytoplasmically exposed regions in Escherichia coli. Two-dimensional electrophoresis of these single chains also reveal multiple banding patterns. The two-dimensional banding patterns described are unaffected by exposure to acidic or basic conditions, increased gel running time in the first dimension, treatment of the proteins with alkaline phosphatase to remove any potential phosphorylation, or preincubation in the presence of iodoacetamide. Multiple forms of recombinant alpha and beta chains were also observed in Tris-glycine-urea gels which merged into a single band in the presence of SDS. In addition, partially fractionated bands from preparative isoelectric focusing gels, when refocused, showed an identical number of multiple spots spanning the same range of isoelectric points. These results together suggest that each polypeptide chain of MHC class II antigens may exist in multiconformational forms, and the observed charge heterogeneity is independent of glycosylation and phosphorylation of the proteins.
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PMID:Intramolecular charge heterogeneity in purified major histocompatibility class II alpha and beta polypeptide chains. 814 5

Nine different isoenzymes and (or) isoforms of alkaline phosphatase (ALP; EC 3.1.3.1) from human tissue were studied with respect to Km and Vmax values for p-nitrophenyl phosphate (p-NPP) in seven different potential phosphoacceptors/buffers. Generally, the phosphoacceptors/buffers with the lowest affinity for p-NPP (highest Km values) gave the highest Vmax values; for the nine enzyme forms in this study, the mean Km and Vmax values were greatest in 2-(ethylamino) (EAE). The two amino-propanol buffers gave the lowest Km and Vmax values. The phosphoacceptors/buffers N-methyl-D-glucamine (MEG), diethanolamine, and Tris had intermediate Km and Vmax values. Hydrophilic liver ALP retained > 90% of its activity after 24 h at 30 degrees C in both 1.0 and 0.3 mol/L Tris and 2-amino-2-methyl-1,3-propanediol and in 0.3 mol/L MEG. This isoenzyme showed greatest inactivation upon prolonged exposure to 1.0 and 0.3 mol/L EAE, the activity at 24 h being approximately 50-66% of that at zero time. p-NPP underwent the greatest spontaneous degradation, approximately 2.5 times that of baseline levels, in 1 mol/L MEG. There was little degradation in all of the buffers tested at 0.3 mol/L or in Tris, EAE, and 2-amino-2-methyl-1-propanol at 1.0 mol/L.
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PMID:Kinetic parameters for the cleaved substrate, and enzyme and substrate stability, vary with the phosphoacceptor in alkaline phosphatase catalysis. 822 23

The characterization of immobilized Escherichia coli alkaline phosphatase reactors used in flow injection analysis is reported for factors such as optimum pH, activity, ionic strength, product inhibition, and substrate specificity. The kinetics of the immobilized enzyme was studied, and mathematical descriptions were developed for the use of an immobilized enzyme packed-bed reactor to evaluate the kinetic parameters and the number of active sites on the immobilized enzyme. Suppression of phosphatase activity by orthophosphate was found to be significantly reduced, and the Michaelis-Menten constant increased when the enzyme was immobilized and packed in a reactor. Immobilized E. coli alkaline phosphatase exhibited similar activity at pH 8 in Tris-HCl, NaHCO3 and borate-HCl buffers but slightly lower activity in NH3H2O-NH4Ac buffer. The performance of the immobilized enzyme reactor was not affected by the presence of up to 10 M Mg(II), Ni(II), Cd(II), Co(II), Mn(II), Cu(II), or urea, 1 M Fe(II), or 0.1 M Fe(III) in the substrate stream. The chelating agent EDTA, however, gradually deactivated the immobilized enzyme. The periodic restoration of enzyme activity was achieved following the removal and addition of zinc ions. The immobilized E. coli alkaline phosphatase packed-bed reactor was used to measure the alkaline phosphatase available phosphorus content of a number of model organophosphorus compounds. p-Nitrophenyl phosphate showed a linear response in the range of 1.6 x 10(-7)-1.6 x 10(-4) M. This study forms part of a larger program to develop enzymatic systems for water quality measurement.
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PMID:Characterization of immobilized Escherichia coli alkaline phosphatase reactors in flow injection analysis. 825 68

The X-ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His-412 was replaced by Asn has been determined at both low (-Zn) and high (+Zn) concentrations of zinc. In the wild-type structure, His-412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn1) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614-31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild-type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N (+Zn) enzyme was refined to an R-factor of 0.174 at 2.2 A resolution, and the structure of the H412N(-Zn) enzyme was refined to an R-factor of 0.166 at a resolution of 2.6 A. Both indicated that the Asn residue substituted for His-412 did not coordinate well to Zn1. In the H412N(-Zn) structure, the Zn1 site had very low occupancy and the phosphate was shifted by 1.8 A from its position in the wild-type structure. The Mg binding site was also affected by the substitution of Asn for His-412. Both structures of the H412N enzyme also revealed a surface-accessible cavity near the Zn1 site that may serve as a binding site for Tris.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Escherichia coli alkaline phosphatase: X-ray structural studies of a mutant enzyme (His-412-->Asn) at one of the catalytically important zinc binding sites. 852 Apr 75

Site-specific mutagenesis has been used to replace His-412 with glutamine in Escherichia coli alkaline phosphatase. In the wild-type enzyme His-412 is a direct ligand to one of the catalytically important zinc atoms (Zn1) in the active site. The mutant enzyme (H412Q) exhibited about the same k(cat), but a 50-fold increase in K(m) compared to the corresponding kinetic parameters for the wild-type enzyme. Furthermore, the H412Q enzyme had a lower zinc content than the wild-type enzyme. In contrast to the wild-type enzyme, Tris was less effective in the transferase reaction and dramatically inhibited the hydrolysis reaction of the H412Q enzyme. The addition of zinc to the mutant enzyme increased the k(cat) value above that of the wild-type enzyme, partially restored the weak substrate and phosphate binding, and also alleviated the inhibition by Tris. The structure of the H412Q enzyme was also determined by X-ray crystallography. The overall structure of the H412Q enzyme was very similar to that of the wild-type enzyme; the only alpha-carbon displacements over 1 angstrom were observed near the mutation site. In the H412Q structure no phosphate was bound in the active site of the enzyme; however, two water molecules were observed where phosphate normally binds in the wild-type enzyme. Close examination of the active site of the H412Q structure revealed structural changes in Ser-102 as well as at the mutation site. For example, the carbonyl oxygen of the side chain of Gln-412 rotated away from the position of His-412 in the wild-type structure, although too far away (3.2 angstroms) to coordinate to Zn1. Studies on the H412Q enzyme, and a comparison of the H412Q and H412N structures, suggest that the structure and electostatics of the imidazole ring of histidine are critical for its function as a zinc ligand in alkaline phosphatase.
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PMID:Kinetic and X-ray structural studies of a mutant Escherichia coli alkaline phosphatase (His-412-->Gln) at one of the zinc binding sites. 865 82

The substitution of aspartate at position 153 in Escherichia coli alkaline phosphatase by glycine results in a mutant enzyme with 5-fold higher catalytic activity (kcat) but no change in Km at pH 8.0 in 50 mM Tris-HCl. The increased kcat is achieved by a faster release of the phosphate product as a result of the lower phosphate affinity. The mutation also affects Mg2+ binding, resulting in an enzyme with lower metal affinity. The 3-D X-ray structure of the D153G mutant has been refined at 2.5 A to a crystallographic R-factor of 16.2%. An analysis of this structure has revealed that the decreased phosphate affinity is caused by an apparent increase in flexibility of the guanidinium side chain of Arg166 involved in phosphate binding. The mutation of Asp153 to Gly also affects the position of the water ligands of Mg2+, and the loop Gln152-Thr155 is shifted by 0.3 A away from the active site. The weaker Mg2+ binding of the mutant compared with the wild type is caused by an altered coordination sphere in the proximity of the Mg2+ ion, and also by the loss of an electrostatic interaction (Mg2+.COO-Asp153) in the mutant. Its ligands W454 and W455 and hydroxyl of Thr155, involved in the octahedral coordination of the Mg2+ ion, are further apart in the mutant compared with the wild type.
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PMID:3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity. 874 24

Sandwich enzyme-linked immunosorbent assay (sELISA) was developed for precise quantitation of cytosolic phospholipase A2 (cPLA2 type IV) concentration in isolated human peripheral blood eosinophils as an alternative to semiquantitative chemiluminescent assay employing immunoprecipitation/Western blot analysis. In this assay, monoclonal mouse anti-human cPLA2 antiserum was used as the capture antibody, polyclonal rabbit anti-human cPLA2 antiserum as the secondary antibody, and alkaline phosphatase-conjugated goat anti-rabbit IgG as the tertiary, reporter antibody. Purified human cPLA2 (0-1000 ng/ml) dissolved in Tris-HCl buffered saline was used as the standard protein. The detection limit for cPLA2 in 10(6) eosinophils was 0.109 ng/ml, and coefficients of inter- and intra-assay variation were 4.23% and 7.07%, respectively. There was no cross-reactivity with other (secretory) isoforms of PLA2 (sPLA2 types I-III) either from porcine pancreas, human synovial fluid, or bee venom. In separate studies, the recovery of cPLA2 was > 83% when eosinophil lysate was supplemented exogenously with two different concentrations of cPLA2. From a total protein content of 22.3 +/- 1.7 micrograms/10(6) cells, the baseline concentration of cPLA2 was 0.38 +/- 0.18 ng/10(6) cells in eosinophils obtained from mildly atopic donors. Immunoblotting studies confirmed the complete specificity for the type IV isoform as detected by sELISA. This sELISA method permits the precise quantitative assessment of cPLA2 in nanogram quantities per million cells, which has not previously been possible by immunoblotting analysis.
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PMID:Quantitation of the cytosolic phospholipase A2 (type IV) in isolated human peripheral blood eosinophils by sandwich-ELISA. 898 53


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