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)

Mutagenesis of the absolutely conserved residue Asp101 of the non-specific monoesterase alkaline phosphatase (E.C. 3.1.3.1) from E. coli has produced an enzyme with increased kcat. The carboxyl group of the Asp101 residue has been proposed to be involved in the positioning of Arg166 and the formation of the helix that contains the active site Ser102. The crystal structure of the Asp101-->Ser mutant has been refined at 2.5 A to a final crystallographic R-factor of 0.173. The altered active site structure of the mutant is compared with that of the wild-type as well as with the structures of the mutant enzyme soaked in two known alkaline phosphatase inhibitors (inorganic phosphate and arsenate). The changes affect primarily the side chain of Arg166 which, by losing the hydrogen bond interaction with the carboxyl side chain of Asp101, becomes more flexible. This analysis, in conjunction with product inhibition studies of the mutant enzyme, suggests that at high pH (> 7) the enzyme achieves a quicker catalytic turnover by allowing a faster release of the product.
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PMID:3-D structure of a mutant (Asp101-->Ser) of E.coli alkaline phosphatase with higher catalytic activity. 148 Jun 14

Alkaline phosphatase was the first zinc enzyme to be discovered in which three closely spaced metal ions (two Zn ions and one Mg ion) are present at the active center. Zn ions at all three sites also produce a maximally active enzyme. These metal ions have center-to-center distances of 3.9 A (Zn1-Zn2), 4.9 A (Zn2-Mg3), and 7.1 A (Zn1-Mg3). Despite the close packing of these metal centers, only one bridging ligand, the carboxyl of Asp51, bridges Zn2 and Mg3. A crystal structure at 2.0-A resolution of the noncovalent phosphate complex, E.P, formed with the active center shows that two phosphate oxygens form a phosphate bridge between Zn1 and Zn2, while the two other phosphate oxygens form hydrogen bonds with the guanidium group of Arg166. This places Ser102, the residue known to be phosphorylated during phosphate hydrolysis, in the required apical position to initiate a nucleophilic attack on the phosphorous. Extrapolation of the E.P structure to the enzyme-substrate complex, E.ROPO4(2-), leads to the conclusion that Zn1 must coordinate the ester oxygen, thus activating the leaving group in the phosphorylation of Ser102. Likewise, Zn2 appears to coordinate the ester oxygen of the seryl phosphate and activate the leaving group during the hydrolysis of the phosphoseryl intermediate. Both of these findings suggest that there may be a significant dissociative character to each of the two displacements at phosphorous catalyzed by alkaline phosphatase. A water molecule (or hydroxide) coordinated to Zn1 following formation of the phosphoseryl intermediate appears to be the nucleophile in the second step of the mechanism. Dissociation of the product phosphate from the E.P intermediate is the slowest, 35 s-1, and therefore the rate-limiting, step of the mechanism at alkaline pH. Since the determination of the initial crystal structure of alkaline phosphatase, two other crystal structures of enzymes involved in phosphate ester hydrolysis have been completed that show a triad of closely spaced zinc ions present at their active centers. These enzymes are phospholipase C from Bacillus cereus (structure at 1.5-A resolution) (43) and P1 nuclease from Penicillium citrinum (structure at 2.8-A resolution) (74). Both enzymes hydrolyze phosphodiesters. Substrates for phospholipase C are phosphatidylinositol and phosphatidylcholine, while P1 nuclease is an endonuclease hydrolyzing single stranded ribo- and deoxyribonucleotides. P1 nuclease also has activity as a phosphomonoesterase against 3'-terminal phosphates of nucleotides. The Zn ions in both enzymes form almost identical trinuclear sites.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structure and mechanism of alkaline phosphatase. 152 73

A series of modified trp operator sequences has been prepared by the incorporation of seven different base analogues. Four of the analogues allow the site-specific deletion of functional groups present on the dA-dT and dT-dA base pairs at positions -4/+4 and -5/+5 in the trp operator. The remaining three analogues permit the incorporation of structural analogues of the native dA-dT or dG-dC base pairs. The duplex operator sequences all exhibit Tm values well above ambient temperature (48-70 degrees C), and these values generally correlate very well with the number of interstrand hydrogen bonds present. The affinity between the trp repressor and 14 modified operator sequences was examined using a recently developed alkaline phosphatase protection assay. The results from the analogue sequences used in this study suggest that the structure of the dA-dT or dT-dA base pairs at positions -4/+4 and -5/+5, respectively, has relatively little effect upon the solution binding by the trp repressor, but the protein is very sensitive to the orientation of the amino and carbonyl functional groups at the -4/+4 positions, which are involved in the formation of an interbase hydrogen bond present in the major groove. (The term structure in this case refers to the hydrogen bonding structure of the base pairs. We recognize that the introduction of conservative functional group deletions or reversals may affect other structural criteria such as hydration.) The deletion of individual functional groups from the operator sequence suggests that the carbonyl at dT+4 is critical for formation of the high-affinity sequence-specific complex. Additionally, the thymine methyl group at dT+4 and the N7 nitrogen of dA+5 appear to be critical contacts necessary for high-affinity binding by the repressor. The thymine carbonyl and the adenine N7 nitrogen are each responsible for approximately -1.5 kcal/mol of apparent free energy of binding. The thymine methyl provides a somewhat smaller contribution of -0.7 kcal/mol. Deletion of either of the adenine amino groups at dA-4 or dA+5 results in a sequence that binds to the repressor with a higher affinity than observed with the native sequence; this can be explained in that the functional groups lost are not critical for binding, and the resulting increased flexibility of the operator, or the creation of a more hydrophobic surface at these sites, enhances van der Waals contacts between the protein and the nucleic acid.
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PMID:Interactions between the trp repressor and its operator sequence as studied by base analogue substitution. 161 Aug 35

The biological function of zinc is governed by the composition of its tetrahedral coordination polyhedron in the metalloprotein, and each ligand group that coordinates to the metal ion does so with a well-defined stereochemical preference. Consequently, protein-zinc recognition and discrimination requires proper chemical composition and proper stereochemistry of the metal-ligand environment. However, it should be noted that the entire protein behaves as the "zinc ligand," since residues that are quite distant from the metal affect recognition and function by through-space (either solvent or the protein milieu) or through-hydrogen bond coulombic interactions. Additionally, long-range interactions across hydrogen bonds serve to orient ligands and therefore minimize the entropy loss incurred on metal binding. Since zinc is not subject to ligand field stabilization effects, it is easy for the tetrahedral protein-binding site to discriminate zinc from other first-row transition metal ions: It is only for Zn2+ that the change from an octahedral to a tetrahedral ligand field is not energetically disfavored. Structural considerations such as these must illuminate the engineering of de novo zinc-binding sites in proteins. Zinc serves chemical, structural, and regulatory roles in biological systems. In biological chemistry zinc serves as an electrophilic catalyst; that is, it stabilizes negative charges encountered during an enzyme-catalyzed reaction. The coordination polyhedron of catalytic zinc is usually dominated by histidine side chains. In biological structure zinc is typically sequestered from solvent, and its coordination polyhedron is almost exclusively dominated by cysteine thiolates. Structural or regulatory zinc is found as either a single metal ion or as part of a cluster of two or more metals. In multinuclear clusters cysteine thiolates either bridge two metal ions or serve as terminal ligands to a single metal ion. Even in complex multinuclear clusters, Zn2+ displays tetrahedral coordination. The structural biology of zinc continues to receive attention in catalytic and regulatory systems such as leucine aminopeptidase, alkaline phosphatase, transcription factors, and steroid receptors. For example, zinc-mediated hormone-receptor association has recently been demonstrated in the binding of human growth hormone to the extracellular binding domain of the human prolactin receptor (Cunningham et al., 1990). To be sure, structural studies of zinc in biology will continue to be a fruitful source of bioinorganic advances, as well as surprises, in the future.
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PMID:Structural biology of zinc. 179 7

Secretory vesicles that accumulate in the temperature-sensitive sec6-4 strain of yeast have been shown to contain a vanadate-sensitive ATPase, presumably en route to the plasma membrane (Walworth, N. C., and Novick, P. J. (1987) J. Cell Biol. 105, 163-174). We have now established this enzyme to be a fully functional form of the PMA1 [H+]ATPase, identical in its catalytic properties to that found in the plasma membrane. In addition, the secretory vesicles are sealed tightly enough to permit the measurement of ATP-dependent proton pumping with fluorescent probes. We have gone on to exploit the vesicles as an expression system for site-directed mutants of the ATPase. For this purpose, a sec6-4 strain has been constructed in which the chromosomal PMA1 gene is under control of the GAL1 promoter; the mutant pma1 allele to be studied is introduced on a centromeric plasmid under the control of a novel heat shock promoter. In galactose medium at 23 degrees C, the wild-type ATPase is produced and supports normal vegetative growth. When the cells are switched to glucose medium at 37 degrees C, however, the wild-type gene turns off, the mutant gene turns on, and secretory vesicles accumulate. The vesicles contain a substantial amount of newly synthesized, plasmid-encoded ATPase (5-10% of total vesicle protein), but only traces of residual wild-type PMA1 ATPase and no detectable mitochondrial ATPase, vacuolar ATPase, or acid or alkaline phosphatase. To test the expression strategy, we have made use of pma1-105 (Ser368----Phe), a vanadate-resistant mutant previously characterized by standard methods (Perlin, D. S., Harris, S. L., Seto-Young, D., and Haber, J. E. (1989) J. Biol. Chem. 264, 21857-21864). In secretory vesicles, as expected, the plasmid-borne pma1-105 allele gives rise to a mutant enzyme with a reduced rate of ATP hydrolysis and a 100-fold increase in Ki for vanadate. Proton pumping is similarly resistant to vanadate. Thus, the vesicles appear well suited for the production and characterization of mutant forms of the PMA1 [H+]ATPase. They should also aid the study of other yeast membrane proteins that are essential for growth as well as heterologous proteins whose appearance in the plasma membrane may be toxic to the cell.
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PMID:Expression of the yeast plasma membrane [H+]ATPase in secretory vesicles. A new strategy for directed mutagenesis. 182 8

High-affinity nickel transport in Alcaligenes eutrophus H16 is mediated by a function designated hoxN. hoxN lies within the hydrogenase gene cluster of megaplasmid pHG1. An insertional mutation at the hoxN locus led to an increased nickel requirement. In this mutant (strain HF260) both autotrophic growth on hydrogen and wild-type level of urease, a nickel-containing enzyme, were dependent on high concentration of nickel in the medium. Studies with a heterologous in vivo expression system revealed that the hoxN locus encodes two proteins with Mr = 30,000 and 28,000. Only the larger polypeptide was essential for nickel transport. The hoxN locus was cloned on a 2.2-kilobase pair fragment. Nucleotide sequence analysis of the hoxN locus revealed an open reading frame with a coding capacity for a protein of 33.1 kDa. The insertion leading to the Nic- phenotype of strain HF260 maps within this open reading frame indicating that it does in fact have coding function. The deduced amino acid sequence of the hoxN gene has several features typical of a hydrophobic integral membrane protein. Alkaline phosphatase fusion proteins produced by insertion of the transposon TnphoA into hoxN gave significant levels of alkaline phosphatase activity indicating that protein HoxN contains periplasmic domains. Taken together, our results suggest that gene hoxN encodes the high-affinity nickel transporter of A. eutrophus.
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PMID:Cloning, nucleotide sequence, and heterologous expression of a high-affinity nickel transport gene from Alcaligenes eutrophus. 184 42

Light-emitting chemical reactions (chemiluminescence, CL) and biological reactions (bioluminescence, BL) have a diverse range of analytical applications but relatively few have been adopted by routine clinical laboratories. Advantages of CL and BL assays include sensitivity (attomole and sub-attomole detection limits), speed (signal generated in a few seconds and in some cases stable for several hours), nonhazardous reagents, and simple procedures. The most promising clinical applications are in immunoassay, protein blotting, and DNA probe assays. Chemiluminescent molecules exploited as labels include luminol, isoluminol, acridinium esters, thioesters and sulfonamides, and phenanthridinium esters. Separation and nonseparation assays have been devised, based on isoluminol and acridinium ester labels. The combination of the amplification properties of an enzyme and a CL or BL detection reaction provides a highly sensitive analytical system. Since 1983, CL and BL methods have been developed for many enzyme labels, e.g., alkaline phosphatase, glucose-6-phosphate dehydrogenase, horseradish peroxidase, Renilla luciferase, and xanthine oxidase. Currently, the most successful enzyme assays are the enhanced CL method for a peroxidase label involving a mixture of luminol, hydrogen peroxide, and an enhancer (e.g., p-iodophenol) and the direct CL method for alkaline phosphatase, with an adamantyl 1,2-dioxetane phenyl phosphate as substrate. Both systems are very sensitive (the detection limit for alkaline phosphatase when using the dioxetane reagent is 0.001 amol) and produce long-lived light emission (greater than 30 min), which is ideal for membrane applications in which light emission is detected with photographic film or a charge-coupled device camera.
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PMID:Chemiluminescent and bioluminescent techniques. 189 71

A highly sensitive flavin adenine dinucleotide-3'-phosphate (FADP)-based enzyme amplification cascade has been developed for determining alkaline phosphatase (ALP; EC 3.1.3.1). The cascade detects ALP via the dephosphorylation of the novel substrate FADP to produce the cofactor FAD, which binds stoichiometrically to inactive apo D-amino acid oxidase (D-AAO). The resulting active holo D-AAO oxidizes D-proline to produce hydrogen peroxide, which is quantified by the horseradish peroxidase-mediated conversion of 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine to a colored product. The FADP-based enzyme amplification cascade has been used in a novel releasable linker immunoassay (RELIA) to quantify thyrotropin (TSH). In the assay, TSH is first captured onto antibody-coated chromium dioxide particles. After formation of an antibody-TSH sandwich with a dethiobiotinylated second antibody, the complex is reacted with a streptavidin-ALP conjugate. Biotin is then used to release the conjugate into solution, and ALP is quantified in an automated version of the FADP-based amplification cascade on the aca discrete clinical analyzer (Du Pont). The sensitivity of the colorimetric RELIA assay for TSH (less than 0.1 milli-int. unit/L) is comparable with that of fluorometric assays. This technology provides a way to adapt to the aca high-sensitivity immunoassays for a wide range of analytes via colorimetric detection.
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PMID:Sensitive, colorimetric enzyme amplification cascade for determination of alkaline phosphatase and application of the method to an immunoassay of thyrotropin. 189 77

Escherichia coli alkaline phosphatase catalyzes the hydrolysis of a wide variety of phosphomonoesters at similar rates, and the reaction proceeds through a phosphoenzyme intermediate. The active site region is highly conserved between the E. coli and mammalian alkaline phosphatases. The three-dimensional structure of the E. coli enzyme indicates that Lys-328, which is replaced by histidine in all mammalian alkaline phosphatases, is bridged to the phosphate through a water molecule. This water molecule is also hydrogen bonded to Asp-327, a bidendate ligand of the one of the two zinc atoms. Here we report the use of site-specific mutagenesis to convert Lys-328 to both histidine and alanine. Steady-state kinetic studies above pH 7.0 indicate that both mutant enzymes have altered pH versus activity profiles compared to the profile for the wild-type enzyme. At pH 10.3, in the presence of Tris, the Lys-328----Ala enzyme is approximately 14-fold more active than the wild-type enzyme. At the same pH in the absence of Tris the Lys-328----Ala enzyme is still 6-fold more active than the wild-type enzyme. Both mutant enzymes have lower phosphate affinities than the wild-type enzyme at all pH values investigated. Pre-steady-state kinetics at pH 5.5 reveal that the Lys-328----Ala enzyme behaves very similar to the phosphate-free wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A water-mediated salt link in the catalytic site of Escherichia coli alkaline phosphatase may influence activity. 190 46

A novel one-step double immunolabeling method was elaborated on the basis of the simultaneous application of preformed molecular complexes of two primary antibodies with their specific secondary antibodies labeled with different enzymes. Treatment with a rat monoclonal antibody (MAb), M1-8, pre-coupled with horseradish peroxidase-linked sheep anti-rat immunoglobulins, and enzyme reaction revealed by the 3-amino-9-ethylcarbazole/hydrogen peroxide reaction, resulted in red-brown intracytoplasmic staining of interdigitating reticular cells in the lymph nodes of Balb/c mice. Another molecular complex, made of mouse anti-Ia MAb with alkaline phosphatase-linked rabbit anti-mouse immunoglobulins, applied at the same time and then developed with naphthol AS-BI-phosphate/fast blue BB as substrate, yielded blue surface staining of this cell type in addition to labeling of B-lymphocytes. The method described provides the possibility of relatively rapid double antigen detection where the binding sites of the secondary antibodies are saturated by the specific primary immunoglobulins. This approach seems to avoid nonspecific binding of primary antibodies to Fc receptors, and the unwanted binding of secondary antibodies with cell surface immunoglobulins on B-lymphocytes or with crossreactive primary antibodies used in the other sequence, if the primary antibodies and the tissue are the same or crossreactive animal species.
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PMID:One-step double immunolabeling of mouse interdigitating reticular cells: simultaneous application of pre-formed complexes of monoclonal rat antibody M1-8 with horseradish peroxidase-linked anti-rat immunoglobulins and of monoclonal mouse anti-Ia antibody with alkaline phosphatase-coupled anti-mouse immunoglobulins. 194 Mar 24


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