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 room-temperature tryptophan (Trp) phosphorescence lifetime is sensitive to details of the local environment and has been shown to increase significantly in some proteins following H-D exchange. Careful analysis of the phosphorescence lifetime distribution of Trp 109 in Escherichia coli alkaline phosphatase (AP) in solution as a function of time during the H-D exchange shows that this process corresponds to a two-state reaction resulting from the deuteration of a single, specific hydrogen in the core of the protein. The absence of a pH dependence of the exchange rate suggests that the exchange is not an EX2 process, and therefore, a certain degree of unfolding is required for exchange to occur. This discovery opens up the use of phosphorescence-detected hydrogen exchange as a sensitive tool for monitoring the local susceptibility and activation energy for exchange in proteins having a phosphorescent Trp and, for example, for studying the effects of local mutations upon that susceptibility.
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PMID:Hydrogen exchange at the core of Escherichia coli alkaline phosphatase studied by room-temperature tryptophan phosphorescence. 1068 27

Cold-adaptation of enzymes involves improvements in catalytic efficiency. This paper describes studies on the conformational stability of a cold-active alkaline phosphatase (AP) from Atlantic cod, with the aim of understanding more clearly its structural stability in terms of subunit dissociation and unfolding of monomers. AP is a homodimeric enzyme that is only active in the dimeric state. Tryptophan fluorescence, size-exclusion chromatography and enzyme activity were used to monitor alterations in conformational state induced by guanidinium chloride or urea. In cod AP, a clear distinction could be made between dissociation of dimers into monomers and subsequent unfolding of monomers (fits a three-state model). In contrast, dimer dissociation of calf AP coincided with the monophasic unfolding curve observed by tryptophan fluorescence (fits a two-state model). The DeltaG for dimer dissociation of cod AP was 8.3 kcal.mol-1, and the monomer stabilization free energy was 2.2 kcal.mol-1, giving a total of 12.7 kcal.mol-1, whereas the total free energy of calf intestinal AP was 17.3 kcal.mol-1. Thus, dimer formation provided a major contribution to the overall stability of the cod enzyme. Phosphate, the reaction product, had the effect of promoting dimer dissociation and stabilizing the monomers. Cod AP has reduced affinity for inorganic phosphate, the release of which is the rate-limiting step of the reaction mechanism. More flexible links at the interface between the dimer subunits may ease structural rearrangements that facilitate more rapid release of phosphate, and thus catalytic turnover.
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PMID:Dissociation and unfolding of cold-active alkaline phosphatase from atlantic cod in the presence of guanidinium chloride. 1102 83

Phosphoribosyl-pyrophosphate synthetase (Prs) catalyses the synthesis of phosphoribosyl pyrophosphate (PRPP), an intermediate in nucleotide metabolism and the biosynthesis of the amino acids histidine and tryptophan. The Saccharomyces cerevisiae genome contains a family of five PRS genes, PRS1-PRS5. Using anti-peptide antisera directed against two different epitopes of Prs1p it was shown that Prs1p localizes to granular cytoplasmic structures. This localization was confirmed by living cell microscopy of strains expressing a functional green fluorescent protein (GFP)-tagged Prs1p. Analysis of Prs1p distribution in conditional secretory-deficient (sec) mutants suggested that the observed distribution of Prs1p is independent of the secretory pathway. Electron microscopy revealed that plasma membrane invaginations and accumulation of cytoplasmic vesicles were more frequent in strains which lack some of the PRS genes than in the wild-type. The fact that Deltaprs1 and Deltaprs3 are hypersensitive to caffeine and unable to recover from exposure to it as judged by the release of alkaline phosphatase points to a possible link between Prs and the maintenance of cell integrity.
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PMID:The importance of the five phosphoribosyl-pyrophosphate synthetase (Prs) gene products of Saccharomyces cerevisiae in the maintenance of cell integrity and the subcellular localization of Prs1p. 1110 85

Our initial studies of hydrogen-deuterium (H-D) exchange of tryptophan 109 in Escherichia coli alkaline phosphatase (AP) suggested that significant local unfolding of the protein might occur to allow for the exchange reaction, which is very slow at room temperature (Fischer et al., Biochemistry 39 (2000) 1455-1461). In order to investigate whether the partial unfolding and/or 'breathing' motions leading to H-D exchange were part of the unfolding pathway of the protein we prepared a series of mutants, designed to produce cavities around the exchanging residue, and compared their rates of H-D exchange to their lability (rate of inactivation) in guanidine hydrochloride (Gd:HCl). The complex unfolding kinetics of the mutants in the presence of Gd:HCl showed several components with rates that differed substantially among these proteins, but none of the rates of denaturation induced with Gd:HCl was consistently correlated with the H-D exchange rates. We conclude that the partial opening of the AP structure during the H-D exchange of tryptophan 109, although very slow, is not a rate determining step in the unfolding of this protein.
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PMID:Differences in the pathways for unfolding and hydrogen exchange among mutants of Escherichia coli alkaline phosphatase. 1134 35

The phosphorescence and zero field optically detected magnetic resonance (ODMR) of the tryptophan (Trp) residues of alkaline phosphatase from Escherechia coli are examined. Each Trp is resolved optically and identified with the aid of the W220Y mutant and the terbium complex of the apoenzyme. Trp(109), known from earlier work to be the source of room-temperature phosphorescence (RTP), emits a highly resolved low-temperature phosphorescence (LTP) spectrum and has the narrowest ODMR bands observed thus far from any protein site, revealing a uniquely homogeneous local environment. The decay kinetics of Trp(109) at 1.2 K reveals that the major triplet population (70%) undergoes inefficient crystallike spin-lattice relaxation by direct interaction with lattice phonons, the remainder being relaxed efficiently by local disorder modes. The latter population is smaller than is typical for protein sites, suggesting an unusual degree of local rigidity and order consistent with the long-lived RTP. Trp(220) emits a broader LTP spectrum originating to the blue of Trp(109). It has typically broad ODMR bands consistent with local heterogeneity. The LTP of Trp(268) has an ill-defined origin blue shifted relative to Trp(220) and ODMR frequencies consistent with a greater degree of solvent exposure. Trp(268) has noticeable dispersion of its decay kinetics, consistent with quenching at the triplet level by a nearby disulfide residue.
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PMID:Characterization of the tryptophan residues of Escherechia coli alkaline phosphatase by phosphorescence and optically detected magnetic resonance spectroscopy. 1173 24

The hyperthermophilic bacterium Thermotoga maritima encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). The putative gene was isolated and the corresponding protein expressed in Escherichia coli, with and without a predicted signal sequence. The recombinant protein showed phosphatase activity toward the substrate p-nitrophenyl-phosphate with a k(cat) of 16 s(-1) and a K(m) of 175 microM at a pH optimum of 8.0 when assayed at 25 degrees C. T. maritima phosphatase activity increased at high temperatures, reaching a maximum k(cat) of 100 s(-1), with a K(m) of 93 microM at 65 degrees C. Activity was stable at 65 degrees C for >24 h and at 90 degrees C for 5 h. Phosphatase activity was dependent on divalent metal ions, specifically Co(II) and Mg(II). Circular dichroism spectra showed that the enzyme gains secondary structure on addition of these metals. Zinc, the most common divalent metal ion required for activity in known APs, was shown to inhibit the T. maritima phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the presence of 0.1 mM EDTA. The T. maritima AP primary sequence is 28% identical when compared with E. coli AP. Based on a structural model, the active sites are superimposable except for two residues near the E. coli AP Mg binding site, D153 and K328 (E. coli numbering) corresponding to histidine and tryptophan in T. maritima AP, respectively. Sucrose-density gradient sedimentation experiments showed that the protein exists in several quaternary forms predominated by an octamer.
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PMID:Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity. 1191 33

Cytochrome (cyt) c forms complexes, undergoes a conformational change and becomes partly reduced at interaction with membrane anchored alkaline phosphatase (AP), a glycoprotein which is released into the body fluid in forms differing in hydrophobicity. The proportion of products formed in the mixtures depends on pH, ionic strength, temperature and the buffer composition. The reaction terminates in an equilibrium between cyt c(FeII) and other cyt c conformers. Optimal conditions for the rate of the reaction are 100 mM glycine/NaOH, pH 9.7-9.9, at which 68-74% of cyt c is found in the reduced state. The interaction affects compactness of the haem cleft as shown by changes induced in CD spectra of the Soret region and changes in optical characteristics of phenylalanine, tyrosine and tryptophan residues. Differential scanning calorimetry of AP+cyt c mixtures revealed a creation of at least two types of complexes. A complex formed by non-coulombic binding prevails at substoichiometric AP/cyt c ratios, at higher ratios more electrostatic attraction is involved and at 1:1 molar ratio an apparent complexity of binding forces occurs. The rapid phase of the cyt c(FeII) formation depends on the presence of the hydrophobic alkylacylphosphoinositol (glycosylphosphatidylinositol) moiety, the protein part of the enzyme participates in an electrostatic and much slower phase of cyt c(FeII) creation. The results show that non-coulombic interaction may participate at interaction of cyt c with cellular proteins.
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PMID:Cytochrome c forms complexes and is partly reduced at interaction with GPI-anchored alkaline phosphatase. 1196 Jun 83

The influence of different denaturants on the phosphorescence spectrum and lifetime decay of Escherichia coli alkaline phosphatase (AP) was investigated. Phosphorescence intensity and lifetime of tryptophan residue (Trp-109) decrease upon addition of guanidine hydrochloride, ethylene diamine tetraacetic acid, and urea or decreasing acidity. The experiments show that AP undergoes different pathways with different denaturants and that the activation energy data, DeltaS degrees (not equal) and deltaH degrees (not equal) further confirm that there is a stable intermediate state between the folded and unfolded AP states in solution.
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PMID:Study on Escherichia coli alkaline phosphatase conformation by phosphorimetry in the presence of denaturant. 1458 94

An inexpensive and quick deoxygenation system for measuring protein phosphorescence is described. Oxygen was first reduced to less than 1 ppb from nitrogen or other inert gas by passing through an oxygen trap. The oxygen-free gas was routed through stainless steel tubing directly into the sample compartment of the phosphorimeter. Flexible tubing, coupled to the stainless steel tubing, was run through the septum of a cuvette sealed with a gray butyl rubber lyophilization stopper. The flexible tubing allowed for manipulation of the cuvette during alternate cycles of vacuuming and nitrogen equilibration. Utility of the system was demonstrated by measuring the phosphorescence lifetimes of N-acetyl-L-tryptophanamide, alkaline phosphatase, human serum albumin, and recombinant human serum albumin. Phosphorescence lifetimes of 2 ms for N-acetyl-L-tryptophanamide, almost double that previously reported, were routinely achieved while a lifetime of 1.84 s was obtained for alkaline phosphatase, well within the reported range of 1.5-2s. Human serum albumin, which contains a single tryptophan, showed a biexponential decay with lifetimes of 4.33 and 17 ms, in contrast to previous reports of a biexponential decay with rates of 0.2 and 0.9 ms. Recombinant human serum albumin was even more striking with lifetimes of 4.60 and 68.2 ms. The data are explained based on the recently published X-ray crystallographic structure of human serum albumin. The simplicity and reproducibility of the system should make this technique practical for most biochemical labs.
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PMID:A deoxygenation system for measuring protein phosphorescence. 1465 52

With the help of the methods of tryptophan fluorescence and room-temperature phosphorescence and using Escherichia coli alkaline phosphatase as an example, the ability of a protein to exist in a manifold of partially folded thermodynamically stable states differing in conformation, the internal dynamics, and functional activity was shown. Such intermediate (between native and unfolded) structures may form during unfolding or folding of the protein. It was shown that the degree of destruction of the native structural organization of the globule depends on both the nature and the mode of action of the destroying agent and the structure of the protein. Conformational transitions of the globule can change the kind of the internal dynamics (fast, slow), and shifts of dynamics can initiate conformation changes of the protein and precede them. A scheme of the structural and functional transformations of the protein during denaturation is presented, which takes into account the possibility of globule transitions into a manifold of functional active and inactive partially folded states. The role of partially folded forms of cell proteins in the development of pathology is discussed.
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PMID:[On the possibility for a protein to exist in a manifold of partially folded states]. 1532


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