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PubMed1989

Photolabeling of the phosphate binding site of mitochondrial F1-ATPase by [32P]azidonitrophenyl phosphate. Identification of the photolabeled amino acid residues.

[32P]Azidonitrophenyl phosphate [( 32P]ANPP) is a photoactivatable analogue of Pi. It competes efficiently with Pi for binding to the F1 sector of beef heart mitochondrial ATPase and photolabels the Pi binding site located in the beta subunit of F1 [Lauquin, G. J. M., Pougeois, R., & Vignais, P. V. (1980) Biochemistry 19, 4620-4626]. By cleavage of the photolabeled beta subunit of F1 with cyanogen bromide, trypsin, and chymotrypsin, bound [32P]ANPP was localized in a fragment spanning Thr 299-Phe 326. By Edman degradation of the radiolabeled tryptic peptide spanning Ile 296-Arg 337, [32P]ANPP was found to be attached covalently by its photoreactive group to Ile 304, Gln 308, and Tyr 311. These results are discussed in terms of a model in which the phosphate group of [32P]ANPP interacts with a glycine-rich sequence of the beta subunit, spanning Gly 156-Lys 162, which is spatially close to the photolabeled Ile 304-Tyr 311 segment of the same subunit.

Affinity Labels↗
PubMed1979

Spectroscopic studies of pyridoxamine (pyridoxine) 5'-phosphate oxidase. Equilibrium dissociation constants and spectra for riboflavin 5'-phosphate and analogues.

Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC 1.4.3.5) has been shown to bind 1 mol of riboflavin 5'-phosphate (FMN) per mol of apoenzyme and is active with or inhibited by numerous FMN analogues [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436--3442]. The KD values and spectra for selected apoenzyme--flavin complexes have been determined and used to elucidate some of the properties of the FMN-binding site of this flavoprotein. Alterations of the pyrimidinoid portion of the flavin ring decrease binding considerably. The absorption spectra for the protein complexes with 3-deaza-FMN and 8-hydroxy-FMN indicate the presence of a dipolar or positively charged protein group near N1 and O2. The substitution of methyl for hydrogen at N3 apparently causes distortion of the interaction between the flavin ring and an active-site aromatic amino acid residue. Although binding is also decreased somewhat by substitutions at postions 8 and 8 alpha, considerable bulk [e.g., 8-(diethylamino)-FMN and 8 alpha-S-(N-acetyl-cysteinyl)-FMN] is accommodated. Hence, this portion of the flavin ring is probably oriented toward, possibly in contact with, solvent, as has been found for the flavodoxins. The importance of optimum interactions between the flavin and the apoprotein is further emphasized by large differences in the activity of flavin analogues that have similar midpoint potentials in solution.

Apoenzymes↗
PubMed1998

Human DNase I contains mannose 6-phosphate and binds the cation-independent mannose 6-phosphate receptor.

DNase I isolated from human urine (hDNase) or expressed in Chinese hamster ovary (CHO) cells contains mannose-phosphorylated oligosaccharides. hDNase binds to a column of immobilized cation-independent mannose 6-phosphate receptor, with the strongest binding exhibited by the protein bearing diphosphorylated oligosaccharides. The binding is inhibited by 5 mM mannose 6-phosphate, and can be prevented by prior treatment of hDNase with alkaline phosphatase. Phosphorylated high-mannose oligosaccharides were observed at both sites of glycosylation in hDNase by high-performance liquid chromatography-mass spectrometry of a tryptic digest. These results indicate that hDNase, though not an acid hydrolase, may enter the lysosomal trafficking pathway, and may have evolved from a lysosomal enzyme.

Amino Acid Sequence↗
PubMed2002

Immobilization of aminothiols on poly(oxyalkylene phosphates). Formation of poly(oxyethylene phosphates)/cysteamine complexes and their radioprotective efficiency.

The necessity to apply near-toxic amounts of radioprotective drugs to achieve adequate protection during radiation treatments represents a major problem in human medicine. One of the promising strategies to suppress the toxicity of these drugs involves their incorporation into biocompatible polymers. In this study cysteamine (Cy) was attached to poly(oxyethylene phosphate), POEP, via an ionic bond. Radioprotection of E. coli B cells by this substance and its acute toxicity on male C57 BL mice were measured. The toxicity of Cy immobilized within the poly(oxyethylene phosphate) was significantly lower in comparison to pure Cy while its radioprotective efficiency remained high at half the maximum tolerable dose. The high radioprotective efficiency of the Cy/POEP complexes was further confirmed on mice at different polymer molecular weight characteristics, drug immobilization degrees, application times, and doses. It was found that POEP with molecular weight 4700 Da and containing 24% repeating units with attached Cy has the highest protection potential combined with a depot effect.

Animals↗
PubMed2005

Synthesis of (+/-)-2-O-[4'-(N-9''-purinyl)butyl] myo-inositol 1,4,5-tris(phosphate), a potent full agonist at the D-myo-inositol 1,4,5-tris(phosphate) receptor.

Racemic 2-O-[4'(9''-N-purinyl)butyl] myo-inositol 1,4,5-tris(phosphate) 8 was synthesized starting from myo-inositol. Substitution of position 2 by an alkyl side chain was rendered possible by inversion of the chair conformation of the inositol ring by means of an orthoester. The final compound is a full agonist with the same order of potency as d-myo-inositol 1,4,5-tris(phosphate).

Adrenal Cortex↗
PubMed2005

Synthesis and evaluation of 1-deoxy-D-xylulose 5-phosphate analogues as chelation-based inhibitors of methylerythritol phosphate synthase.

[structures: see text] A series of 1-deoxy-D-xylulose 5-phosphate (DXP) analogues were synthesized and evaluated as inhibitors of E. coli methylerythritol phosphate (MEP) synthase. In analogues 1-4, the methyl group in DXP was replaced by hydroxyl, hydroxylamino, methoxy, and amino moieties, respectively. In analogues 5 and 6, the acetyl moiety in DXP was replaced by hydroxymethyl and aminomethyl groups. These compounds were designed to coordinate to the active site divalent metal in MEP synthase. The carboxylate (1), methyl ester (3), amide (4), and alcohol (5) analogues were inhibitors with IC50's ranging from 0.25 to 1.0 mM. The hydroxamic acid (2) and amino (6) analogues did not inhibit the enzyme.

Aldose-Ketose Isomerases↗
PubMed2006

Lewis acid mediated regioselective ring opening of benzylglycidol with dibenzyl phosphate: Short and attractive synthesis of dihydroxyacetone phosphate.

A novel, mild, and efficient method was described to introduce a dibenzyl phosphate by ring opening of benzylglycidol mediated by Lewis acids. This methodology was used as a key step for synthesizing the dihydroxyacetone phosphate (DHAP) in only three steps with an overall yield of 74% from the commercially available racemic benzylglycidol.

Journal Article↗
PubMed2002

Synthesis of a layered zinc phosphate, [NH3(CH2)2NH2(CH2)3NH3][Zn2(PO4)(HPO4)2].H2O, and its transformation to a extra-large pore three-dimensional zinc phosphate, [NH3(CH2)2NH2(CH2)3NH3][Zn3(PO4)(HPO4)3].

An unusual two-dimensional zinc phosphate with pendant phosphate groups, projecting into the inter-lamellar space between the layers, has been synthesized and is shown to transform into a three-dimensional structure with 16-membered bifurcated channels, giving evidence for the building up process in the formation of open-framework structures.

Journal Article↗
PubMed2003

Rapid and flexible synthesis of 1-deoxy-D-xylulose-5-phosphate, the substrate for 1-deoxy-D-xylulose-5-phosphate reductoisomerase.

1-Deoxy-D-xylulose-5-phosphate (DXP) is a key intermediate in the non-mevalonate pathway to terpenoids in bacteria, and it is the substrate for the enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXP-R). In order to study the mechanism of DXP-R, we required a flexible synthesis of the substrate which would allow the incorporation of isotopic labels, and the variation of the two stereocentres. Thus 1,4-dihydroxypent-2-yne was selectively reduced to give the E-olefin, and selective phosphorylation of the primary alcohol followed by oxidation of the secondary alcohol gave a substrate suitable for dihydroxylation. Dihydroxylation using stoichiometric OsO4 in the presence of chiral ligands gave protected DXP in high ee. Final hydrogenolysis gave DXP in quantitative yield and high purity. DXP-R was produced by rapid cloning of the dxr gene from Escherichia coli through controlled expression and ion exchange chromatography. The synthetic DXP was fully active in enzyme assays catalysed by recombinant DXP-R.

Alcohols↗
PubMed1972

The stereochemical structure of disodium DL-glycerol 3-phosphate hexahydrate, the D isomer of which is an inhibitor of triose phosphate isomerase.

The stereochemistry of dl-glycerol 3-phosphate was studied by X-ray-crystallographic techniques. All the bond lengths and angles are within normally accepted limits except the ester bond, which is one of the largest yet noted, being 0.1637nm. The conformation of the molecule is such that an intramolecular hydrogen bond is formed between the hydroxyl group on the beta-carbon atom and the phosphate group. The crystal, which was grown by alcohol diffusion into an aqueous solution, is held together by sodium co-ordination and a complex system of hydrogen bonds. A table of the observed and calculated structure factors, F(obs.) and F(calc.), has been deposited as Supplementary Publication 50010 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1972) 126, 5.

Carbohydrate Epimerases↗
PubMed2005

Regulation of cell survival by lipid phosphate phosphatases involves the modulation of intracellular phosphatidic acid and sphingosine 1-phosphate pools.

We have shown previously that LPPs (lipid phosphate phosphatases) reduce the stimulation of the p42/p44 MAPK (p42/p44 mitogen-activated protein kinase) pathway by the GPCR (G-protein-coupled receptor) agonists S1P (sphingosine 1-phosphate) and LPA (lysophosphatidic acid) in serum-deprived HEK-293 cells [Alderton, Darroch, Sambi, McKie, Ahmed, N. J. Pyne and S. Pyne (2001) J. Biol. Chem. 276, 13452-13460]. In the present study, we now show that this can be blocked by pretreating HEK-293 cells with the caspase 3/7 inhibitor, Ac-DEVD-CHO [N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde)]. Therefore LPP2 and LPP3 appear to regulate the apoptotic status of serum-deprived HEK-293 cells. This was supported further by: (i) caspase 3/7-catalysed cleavage of PARP [poly(ADP-ribose) polymerase] was increased in serum-deprived LPP2-overexpressing compared with vector-transfected HEK-293 cells; and (ii) serum-deprived LPP2- and LPP3-overexpressing cells exhibited limited intranucleosomal DNA laddering, which was absent in vector-transfected cells. Moreover, LPP2 reduced basal intracellular phosphatidic acid levels, whereas LPP3 decreased intracellular S1P in serum-deprived HEK-293 cells. LPP2 and LPP3 are constitutively co-localized with SK1 (sphingosine kinase 1) in cytoplasmic vesicles in HEK-293 cells. Moreover, LPP2 but not LPP3 prevents SK1 from being recruited to a perinuclear compartment upon induction of PLD1 (phospholipase D1) in CHO (Chinese-hamster ovary) cells. Taken together, these data are consistent with an important role for LPP2 and LPP3 in regulating an intracellular pool of PA and S1P respectively, that may govern the apoptotic status of the cell upon serum deprivation.

Animals↗
PubMed2003

Xylulose 5-phosphate mediates glucose-induced lipogenesis by xylulose 5-phosphate-activated protein phosphatase in rat liver.

Carbohydrate-responsive element binding protein (ChREBP) is a transcription factor that activates lipogenic genes in liver in response to excess carbohydrate in the diet. ChREBP is regulated in a reciprocal manner by glucose and cAMP. cAMP-dependent protein kinase (protein kinase A) phosphorylates two physiologically important sites in ChREBP, Ser-196, which is located near nuclear localization signal sequence (NLS), and Thr-666, within the basic helix-loop-helix (bHLH) site, resulting in inactivation of nuclear translocation of ChREBP and of the DNA-binding activity, respectively. We demonstrate here that crude cytosolic extracts from livers of rats fed a high carbohydrate diet contained protein phosphatase (PPase) activity that dephosphorylated a peptide containing Ser-196, whereas a PPase in the nuclear extract catalyzed dephosphorylation of Ser-568 and Thr-666. All these PPases are activated specifically by xylulose 5-phosphate (Xu5P), but not by other sugar phosphates. Furthermore, addition of Xu5P elevated PPase activity to the level observed in extracts of fed liver cells. These partially purified PPases were characterized as PP2A-AB delta C by immunoblotting with specific antibodies. These results suggest that (ia) Xu5P-dependent PPase is responsible for activation of transcription of the L-type pyruvate kinase gene and lipogenic enzyme genes, and (ii) Xu5P is the glucose signaling compound. Thus, we propose that the same Xu5P-activated PPase controls both acute and long-term regulation of glucose metabolism and fat synthesis.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of a novel xylulose 5-phosphate-activated protein phosphatase catalyzing dephosphorylation of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase.

We have shown previously (Nishimura, M., Fedorov, S., and Uyeda, K. (1994) (J. Biol. Chem. 269, 26100-26106) that the administration of high concentrations of glucose stimulates dephosphorylation of Fru-6-P,2-kinase: Fru-2,6-bisphosphatase in perfused liver, and xylulose (Xu) 5-P activates the dephosphorylation reaction. To characterize the protein phosphatase, we have purified the Xu 5-P-activated protein phosphatase to homogeneity from livers of rats injected with high glucose. Several protein phosphatases in the livers were separated by DEAE-cellulose chromatography, but only one peak of the enzyme was activated by Xu 5-P. The protein phosphatase was inhibited by okadaic acid (IC50 = 1-3 nM) and did not require Mg2+ or Ca2+, suggesting that the enzyme was type 2A. The enzyme was a heterotrimer (M(r) = 150,000) and consisted of structural (A, 65 kDa) catalytic (C, 36 kDa), and regulatory (B, 52 kDa) subunits. Amino acid sequences of five tryptic peptides derived from the B subunit showed similarity with those of the B alpha isoform of rat protein phosphatase 2A, but five out of 73 residues were different. The protein phosphatase catalyzed dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase, phosphorylase alpha, and pyruvate kinase, and the Km values were 0.8 microM, 3.7 microM, and 2.2 microM, respectively. Among these substrates dephosphorylation of only the bifunctional enzyme was activated by Xu 5-P, and the K alpha value for Xu 5-P was 20 microM. Xu 5-P was the only sugar phosphate which activated the PP2A among all the sugar phosphates examined. These results demonstrated the existence and isolation of a unique heterotrimeric protein phosphatase 2A in rat liver which catalyzed the dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase and was activated specifically by Xu 5-P. The Xu 5-P-activated protein phosphatase 2A explains the increased Fru 2,6-P2 level in liver after high glucose administration.

Amino Acid Sequence↗
PubMed1998

The two mannose 6-phosphate binding sites of the insulin-like growth factor-II/mannose 6-phosphate receptor display different ligand binding properties.

The two mannose 6-phosphate (Man-6-P) binding sites of the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) have been localized to domains 1-3 and 7-9, and studies have shown that Arg435 in domain 3 and Arg 1334 in domain 9 are essential for Man-6-P binding. To determine whether the IGF-II/MPR containing a single Man-6-P binding site is functional, clonal mouse L cell lines stably transfected with either mutant bovine IGF-II/MPR cDNA, containing substitutions at position 435 and/or 1334, or the wild type receptor cDNA were assayed for their ability to sort lysosomal enzymes to the lysosome. Mutant receptors containing a single Man-6-P binding site were approximately 50% less efficient than the wild type receptor in the overall targeting of lysosomal enzymes to the lysosome. Mutant receptors containing a substitution at Arg1334 (Dom9(Ala)), in contrast to those containing a substitution at Arg435 (Dom3(Ala)), were unable to target cathepsin D and beta-hexosaminidase to the lysosome. Equilibrium binding assays using 125I-labeled beta-glucuronidase demonstrated that Dom3(Ala) and Dom9(Ala) had a Kd of 2.0 and 4.3 nM, respectively. In addition, Dom3(Ala), unlike Dom9(Ala), was unable to completely dissociate from ligand under acidic pH conditions. These data indicate that the two Man-6-P binding sites of the IGF-II/MPR are not functionally equivalent.

Animals↗
PubMed1999

Crystal structure of the H256A mutant of rat testis fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities.

Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P, 2-kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the interconversion of beta-D-fructose- 6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru-2,6-P2) at distinct active sites. A mutant rat testis isozyme with an alanine replacement for the catalytic histidine (H256A) in the Fru-2,6-Pase domain retains 17% of the wild type activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166-2175). We have solved the crystal structure of H256A to a resolution of 2. 4 A by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the important interactions in substrate/product binding. A superposition of the H256A structure with the RT2K-Wo structure reveals no significant reorganization of the active site resulting from the binding of Fru-6-P or the H256A mutation. Using this superposition, we have built a view of the Fru-2,6-P2-bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechanisms for the wild type and mutant proteins. The wild type mechanism would lead to an inefficient transfer of a proton to the leaving group Fru-6-P, which is consistent with a view of this event being rate-limiting, explaining the extremely slow turnover (0. 032 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.

Amino Acid Sequence↗
PubMed2000

Histidine 268 in 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase plays the same role as histidine 202 in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase.

The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (Phe) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first order with respect to enzyme and DEPC concentrations with a pseudo-second order rate constant of inactivation by DEPC of 4.9 +/- 0.8 m(-1) s(-1) at pH 6.8 and 4 degrees C. The dependence of inactivation on pH and the spectral features of enzyme modified at specific pH values imply that both histidine and cysteine residues are modified, which is confirmed by site-directed mutagenesis. Analysis of the chemical modification data indicates that one histidine is essential for activity. DAH 7-P synthase (Phe) is protected against DEPC inactivation by phosphoenolpyruvate, whereas d-erythrose 4-phosphate offers only minimal protection. The conserved residues H-172, H-207, H-268, and H-304 were individually mutated to glycine. The H304G and H207G mutants retain some level of activity, whereas the H268G and H172G mutants are virtually inactive. A comparison of the circular dichroism spectra of wild-type enzyme and the various mutants demonstrates that H-172 may play a structural role. Comparison of the UV spectra of the H268G and wild-type enzymes saturated with Cu(2+) indicates that the metal-binding site of the H268G mutant resembles that of the wild-type enzyme. The residue H-268 may play a catalytic role based on the site-directed mutagenesis and spectroscopic studies. Cysteine 61 appears to influence the pK(a) of H-268 in the wild-type enzyme. The pK(a) of H-268 increases from 6.0 to 7.0 following mutation of C-61 to glycine.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1982

Inhibition of guanylate cyclase by guanosine 3'-phosphate and 2'-deoxyguanosine 3'-phosphate in silkworm fat body.

Guanosine 3'-phosphate and 2'-deoxyguanosine 3'-phosphate are potent inhibitors of guanylate cyclase of various tissues of the silkworm, Bombyx mori. The inhibitory activity is specific for guanine 3'-nucleotides. Thus, 2'-GMP and 5'-GMP are one order or more less potent than 3'-GMP, and the other 3'-nucleotides except 3'-IMP, i.e. 3'-AMP, 2'-deoxy-3'-AMP, 3'-CMP, and 3'-TMP, have no inhibitory activity. Guanosine and 2'-deoxyguanosine are only weakly active. The 3'-nucleotide inhibition is dependent on the concentration of Mn2+. The concentrations of 2'-deoxy-3'-GMP and 3'-GMP required for 50% inhibition are 0.12 and 0.15 mM, respectively, with 5 mM Mn2+.

Adipose Tissue↗
PubMed2006

Hydrogen phosphate and dihydrogen phosphate salts of 4-aminoazobenzene.

4-Amino-trans-azobenzene {or 4-[(E)-phenyldiazenyl]aniline} can form isomeric salts depending on the site of protonation. Both orange bis{4-[(E)-phenyldiazenyl]anilinium} hydrogen phosphate, 2C12H12N3+.HPO4(2-), and purple 4-[(E)-phenyldiazenyl]anilinium dihydrogen phosphate phosphoric acid solvate, C12H12N3+.H2PO4-.H3PO4, (II), have layered structures formed through O-H...O and N-H...O hydrogen bonds. Additionally, azobenzene fragments in (I) are assembled through C-H...pi interactions and in (II) through pi-pi interactions. Arguments for the colour difference are tentatively proposed.

Journal Article↗