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)

To investigate the role of magnesium at the M3 site in Escherichia coli alkaline phosphatase, site-specific mutagenesis was used to substitute Glu-322, a ligand of the Mg2+ with either aspartic acid (E322D) or alanine (E322A). The residual Mg2+ content of the E322D enzyme is about 16-fold lower than that of the wild-type enzyme, and both mutant enzymes exhibit extremely poor catalytic activity compared to the wild-type enzyme. Mg2+ is a strong activator of the E322D enzyme. The hydrolysis activity of the E322D enzyme maximally stimulated by Mg2+ is 60% of that of the wild-type enzyme. Under conditions that measure the sum of hydrolysis and transphosphorylation activities, the kcat of the E322D enzyme in the presence of 500 mM Mg2+ is 2.6-fold higher than the kcat observed for the wild-type enzyme. Zn2+ also activates the E322D enzyme, although it is not as strong an activator as Mg2+. Competition experiments suggest that the activation of the E322D enzyme by Mg2+ and Zn2+ results from binding of either of these metals to the same site on the enzyme. High concentrations of the substrate p-nitrophenyl phosphate inhibit the activity of the E322D enzyme; however, high concentrations of Mg2+ can overcome this inhibition. Stopped-flow experiments indicate that the rate-limiting step of the nonstimulated E322D enzyme at pH 8.0 differs from that of the wild-type enzyme and involves the breaking of the covalent bond between the enzyme and phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding of magnesium in a mutant Escherichia coli alkaline phosphatase changes the rate-determining step in the reaction mechanism. 810 81

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

We have discovered a single homoallelic nucleotide substitution as the putative cause of the perinatal (lethal) form of hypophosphatasia in Canadian Mennonites. Previous linkage and haplotype analysis in this population suggested that a single mutational event was responsible for this autosomal recessive form of hypophosphatasia. The mutation is a guanosine-to-adenosine substitution at nucleotide position 1177 in exon 10 of the tissue nonspecific (liver/bone/kidney) alkaline phosphatase gene. This Gly317-->Asp mutation segregates exclusively with the heterozygote phenotype we previously assigned by biochemical testing (maximum combined lod score of 18.24 at theta = 0.00). This putative disease-causing mutation has not been described in controls nor in other non-Mennonite probands with both lethal and nonlethal forms of hypophosphatasia studied to date. This Gly317-->Asp mutation changes a polar glycine to an acidic aspartate at amino acid position 317 within the highly conserved active site region of the 507-amino-acid polypeptide. Carrier screening for this lethal mutation in our high-risk population is now feasible.
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PMID:A homoallelic Gly317-->Asp mutation in ALPL causes the perinatal (lethal) form of hypophosphatasia in Canadian mennonites. 840 53

The replacement of aspartic acid by histidine at position 153 in Escherichia coli alkaline phosphatase results in a mutant enzyme that is remarkably similar to certain mammalian alkaline phosphatases that are activated by magnesium in a time-dependent fashion. These mammalian alkaline phosphatases have histidine at the position corresponding to 153 of the E. coli sequence. Here we report the three-dimensional structure of the mutant E. coli alkaline phosphatase with histidine at position 153. The structure reveals that the octahedral magnesium binding site has been converted to a tetrahedral zinc binding site with an imidazole ring nitrogen of His-153 as one of the ligands to the zinc. The alteration in metal binding caused by the mutation could explain the origin of the magnesium activation observed with the mammalian alkaline phosphatases. The structure also reveals differences in the mode of phosphate binding, explaining the enhanced phosphate affinity and the reduced activity of the mutant enzyme in the presence of zinc.
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PMID:Conversion of a magnesium binding site into a zinc binding site by a single amino acid substitution in Escherichia coli alkaline phosphatase. 840 98

Site-specific mutagenesis was used to explore the roles of the side chains of residues Lys-328 and Asp-153 in Escherichia coli alkaline phosphatase. The D153H enzyme exhibits a 3.5-fold decrease in activity at pH 8.0 compared to that of the wild-type enzyme, while a double mutant D153H/K328H exhibits a 16-fold decrease in activity under these conditions. However, the Km values for both enzymes, employing the substrate p-nitrophenyl phosphate, are lower than the value for the wild-type enzyme. The Ki for phosphate, which is pH- and Mg(2+)-dependent, is decreased for the D153H enzyme and increased for the D153H/K328H enzyme. Relative to the wild-type enzyme, both mutant enzymes bind Mg2+ more weakly and undergo a time-dependent activation induced by Mg2+. The half-time of the activation process is independent of the Mg2+ concentration, indicating that the activation most probably involves a conformational change. The pH versus activity profiles of both enzymes are altered relative to that of the wild-type enzyme and exhibit greatly enhanced activity, relative to that of the wild-type enzyme, at high pH values. The pre-steady-state kinetics for the D153H and D153H/K328H enzymes exhibit a transient burst of product formation at pH 8.0, under conditions at which the wild-type enzyme exhibits no transient burst, indicating that at pH 8.0 the hydrolysis of the covalent enzyme-phosphate complex is rate-determining and not the release of phosphate from the noncovalent enzyme-phosphate complex as is observed for the wild-type enzyme. Therefore, these mutations are directly influencing catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Magnesium in the active site of Escherichia coli alkaline phosphatase is important for both structural stabilization and catalysis. 843 39

The binding interactions between platelet fibrinogen receptor, glycoprotein (GP) IIb-IIIa, and kistrin, a snake venom disintegrin protein that contains the adhesion site recognition sequence Arg-Gly-Asp (RGD) and potently inhibits platelet aggregation, have been investigated by site-directed mutagenesis of a synthetic kistrin gene. Kistrin was expressed as a fusion protein in Escherichia coli under control of the alkaline phosphatase promoter. This construction included the stII signal sequence to direct secretion to the periplasmic space and one synthetic (Z) domain of Staphylococcal protein A to allow affinity purification using IgG Sepharose. Kistrin was cleaved from the Z-domain by site-specific proteolysis using a mutant subtilisin BPN' and purified by reverse-phase HPLC. This approach facilitated the rapid purification of a set of 43 alanine replacement mutants whose relative affinity for GP IIb-IIIa was measured by competition with immobilized kistrin and by inhibition of platelet aggregation in human platelet-rich plasma. Alanine replacements at R49, G50, and D51 led to weaker inhibitors of platelet aggregation by 90-fold, 2-fold, and > 200-fold, respectively. The conservative D51E mutant was still > 100-fold less potent whereas R49K had a minor effect (1.8-fold), implying the critical nature of the aspartate for high affinity binding. However, mutations outside of the RGD region led to proteins indistinguishable from kistrin, suggesting no substantial secondary binding interactions. Furthermore, reduced kistrin is not active. We therefore propose that a favorable conformation of the RGD region alone is responsible for the high affinity binding of kistrin to GP IIb-IIIa.
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PMID:Binding interactions of kistrin with platelet glycoprotein IIb-IIIa: analysis by site-directed mutagenesis. 845 99

Prothymosin alpha is post-translationally modified. When human myeloma cells were metabolically labeled with [32P]orthophosphoric acid, they synthesized [32P]prothymosin alpha. The incorporated radioactivity was resistant to DNase and RNases A, T1, and T2, but could be completely removed by alkaline phosphatase. No evidence was found for an RNA adduct as postulated by Vartapetian et al. [Vartapetian, A., Makarova, T., Koonin, E. V., Agol, V. I., & Bogdanov, A. (1988) FEBS Lett. 232, 35-38]. Thin-layer electrophoresis of partially hydrolyzed [32P]prothymosin alpha indicated that serine residues were phosphorylated. Analysis of peptides derived from bovine prothymosin alpha and human [32P]prothymosin alpha by treatment with endoproteinase Lys-C revealed that the amino-terminal 14-mer, with serine residues at positions 1, 8, and 9, was phosphorylated at a single position. Approximately 2% of the peptide in each case contained phosphate. Further digestion of the phosphopeptide with Asp-N followed by C18 reversed-phase column chromatography produced two peptides: a phosphate-free 9-mer containing amino acids 6-14 and a labeled peptide migrating slightly faster than the N-terminal 5-mer derived from the unmodified 14-mer. Positive identification of the phosphorylated amino acid was obtained by colliding the 14-residue phosphopeptide with helium in the mass spectrometer and finding phosphate only in a nested set of phosphorylated fragments composed of the first three, four, and five amino acids. The results prove that prothymosin alpha contains N-terminal acetylserine phosphate. In a synchronized population of human myeloma cells, phosphorylation occurred throughout the cell cycle. Furthermore, prothymosin alpha appeared to be stable, with a half-life slightly shorter than the generation time. Although prothymosin alpha is known to be essential for cell division, the constancy of both the amount of the protein and the degree of its phosphorylation suggests that prothymosin alpha does not directly govern mitosis.
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PMID:Phosphorylation of human and bovine prothymosin alpha in vivo. 848 35

The mechanism by which yeast dipeptidyl aminopeptidase (DPAP) A, type II integral membrane protein, is retained in the late Golgi apparatus has been investigated. Prior work demonstrated that the 118-amino acid cytoplasmic domain is both necessary and sufficient for Golgi retention and that mutant or overexpressed DPAP A no longer retained in the Golgi was delivered directly to the vacuolar membrane (Roberts, C. J., S. F. Nothwehr, and T. H. Stevens. 1992. J. Cell Biol. 119:69-83). Replacement of the DPAP A transmembrane domain with a synthetic hydrophobic sequence did not affect either Golgi retention of DPAP A or vacuolar delivery of the retention-defective form of DPAP A. These results indicate that the DPAP A transmembrane domain is not involved in either Golgi retention or targeting of this membrane protein. A detailed mutational analysis of the cytoplasmic domain of DPAP A indicated that the most important elements for retention were within the eight residue stretch 85-92. A 10-amino acid region from DPAP A (81-90) was sufficient for Golgi retention of alkaline phosphatase, a type II vacuolar membrane protein. Detailed mutational analysis within this 10-amino acid sufficient region demonstrated that a Phe-X-Phe-X-Asp motif was absolutely required for efficient retention. The efficiency of Golgi retention via the DPAP A signal could be diminished by overexpression of wild type but not retention-defective versions of Kex2p, another late Golgi membrane protein, suggesting that multiple Golgi membrane proteins may be retained by a common machinery. These results imply a role for a cytoplasmic signal involving aromatic residues in retention of late Golgi membrane proteins in the yeast Saccharomyces cerevisiae.
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PMID:Membrane protein retention in the yeast Golgi apparatus: dipeptidyl aminopeptidase A is retained by a cytoplasmic signal containing aromatic residues. 850 44

The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not inhibit nodule formation. We conclude that osteoblasts interact with the central cell-binding domain of endogenously produced fibronectin during early stages of differentiation, and that these interactions regulate both normal morphogenesis and gene expression.
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PMID:Fibronectin regulates calvarial osteoblast differentiation. 879 25

The structure and function of the polyamine transport protein PotE was studied. Uptake of putrescine by PotE was dependent on the membrane potential. In contrast, the putrescine-ornithine antiporter activity of PotE studied with inside-out membrane vesicles was not dependent on the membrane potential (Kashiwagi, K., Miyamoto, S., Suzuki, F., Kobayashi, H., and Igarashi, K. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4529-4533). The Km values for putrescine uptake and for putrescine-ornithine antiporter activity were 1.8 and 73 microM, respectively. Uptake of putrescine was inhibited by high concentrations of ornithine. This effect of ornithine appears to be due to putrescine-ornithine antiporter activity because it occurs only after accumulation of putrescine within cells and because ornithine causes excretion of putrescine. Thus, PotE can function not only as a putrescine-ornithine antiporter to excrete putrescine but also as a putrescine uptake protein. Both the NH2 and COOH termini of PotE were located in the cytoplasm, as determined by the activation of alkaline phosphatase and beta-galactosidase by various PotE-fusion proteins. The activities of putrescine uptake and excretion were studied using mutated PotE proteins. It was found that glutamic acid 207 was essential for both the uptake and excretion of putrescine by the PotE protein and that glutamic acids 77 and 433 were also involved in both activities. These three glutamic acids are located on the cytoplasmic side of PotE, and the function of these three residues could not be replaced by other amino acids. Putrescine transport activities did not change significantly with mutations at the other 13 glutamic acid or aspartic acid residues in PotE.
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PMID:Excretion and uptake of putrescine by the PotE protein in Escherichia coli. 904 51


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