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1.
Commun Biol ; 5(1): 272, 2022 03 28.
Article in English | MEDLINE | ID: mdl-35347220

ABSTRACT

Burkholderia pseudomallei lethal factor 1 (BLF1) exhibits site-specific glutamine deamidase activity against the eukaryotic RNA helicase, eIF4A, thereby blocking mammalian protein synthesis. The structure of a complex between BLF1 C94S and human eIF4A shows that the toxin binds in the cleft between the two RecA-like eIF4A domains forming interactions with residues from both and with the scissile amide of the target glutamine, Gln339, adjacent to the toxin active site. The RecA-like domains adopt a radically twisted orientation compared to other eIF4A structures and the nature and position of conserved residues suggests this may represent a conformation associated with RNA binding. Comparison of the catalytic site of BLF1 with other deamidases and cysteine proteases reveals that they fall into two classes, related by pseudosymmetry, that present either the re or si faces of the target amide/peptide to the nucleophilic sulfur, highlighting constraints in the convergent evolution of their Cys-His active sites.


Subject(s)
Burkholderia , Eukaryotic Initiation Factor-4A , Amides , Animals , Burkholderia/genetics , Burkholderia/metabolism , Eukaryotic Initiation Factor-4A/metabolism , Glutamine/metabolism , Humans , Mammals , Protein Biosynthesis
2.
Dokl Biochem Biophys ; 475(1): 245-249, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28864894

ABSTRACT

It is proposed to perform quantum mechanical/molecular dynamics calculations of chemical reactions that are planned to be catalyzed by antibodies and then conduct a virtual screening of the library of potential antibody mutants to select an optimal biocatalyst. We tested the effectiveness of this approach by the example of hydrolysis of organophosphorus toxicant paraoxon using kinetic approaches and X-ray analysis of the antibody biocatalyst designed de novo.


Subject(s)
Antibodies/genetics , Antibodies/metabolism , Biocatalysis , Computational Biology/instrumentation , Mutation , Antibodies/chemistry , Models, Molecular , Protein Conformation
3.
Biochemistry (Mosc) ; 77(10): 1083-96, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23157289

ABSTRACT

Early studies on chemical synthesis of biological molecules can be seen to progress to preparation and biological evaluation of phosphonates as analogues of biological phosphates, with emphasis on their isosteric and isopolar character. Work with such mimics progressed into structural studies with a range of nucleotide-utilising enzymes. The arrival of metal fluorides as analogues of the phosphoryl group, PO(3)(-), for transition state (TS) analysis of enzyme reactions stimulated the symbiotic deployment of (19)F NMR and protein crystallography. Characteristics of enzyme transition state analogues are reviewed for a range of reactions. From the available MF(x) species, trifluoroberyllate gives tetrahedral mimics of ground states (GS) in which phosphate is linked to carboxylate and phosphate oxyanions. Tetrafluoroaluminate is widely employed as a TS mimic, but it necessarily imposes octahedral geometry on the assembled complexes, whereas phosphoryl transfer involves trigonal bipyramidal (tbp) geometry. Trifluoromagnesate (MgF(3)(-)) provides the near-ideal solution, delivering tbp geometry and correct anionic charge. Some of the forty reported tbp structures assigned as having AlF(3)(0) cores have been redefined as trifluoromagnesate complexes. Transition state analogues for a range of kinases, mutases, and phosphatases provide a detailed description of mechanism for phosphoryl group transfer, supporting the concept of charge balance in their TS and of concerted-associative pathways for biocatalysis. Above all, superposition of GS and TS structures reveals that in associative phosphoryl transfer, the phosphorus atom migrates through a triangle of three, near-stationary, equatorial oxygens. The extension of these studies to near attack conformers further illuminates enzyme catalysis of phosphoryl transfer.


Subject(s)
Biocatalysis , Phosphorus/chemistry , Ligands , Organophosphonates/chemistry
4.
J Mol Biol ; 313(1): 111-22, 2001 Oct 12.
Article in English | MEDLINE | ID: mdl-11601850

ABSTRACT

In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing. The autoinducer-2 production protein LuxS, is involved in a novel quorum-sensing system and is thought to catalyse the degradation of S-ribosylhomocysteine to homocysteine and the autoinducer molecule 4,5-dihydroxy-2,3-pentadione. The crystal structure of Bacillus subtilis LuxS has been determined at 1.2 A resolution, together with the binary complexes of LuxS with S-ribosylhomocysteine and homocysteine to 2.2 and 2.3 A resolution, respectively. These structures show that LuxS is a homodimer with an apparently novel fold based on an eight-stranded beta-barrel, flanked by six alpha-helices. Each active site contains a zinc ion coordinated by the conserved residues His54, His58 and Cys126, and includes residues from both subunits. S-ribosylhomocysteine binds in a deep pocket with the ribose moiety adjacent to the enzyme-bound zinc ion. Access to the active site appears to be restricted and possibly requires conformational changes in the protein involving the movement of residues 125-129 and those at the N terminus. The structure contains an oxidised cysteine residue in the active site whose role in the biological process of LuxS has not been determined. The autoinducer-2 signalling pathway has been linked to aspects of bacterial virulence and pathogenicity. The structural data on LuxS will provide opportunities for targeting this enzyme for the rational design of new antibiotics.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Amino Acid Sequence , Binding Sites , Carbon-Sulfur Lyases , Crystallography, X-Ray , Homocysteine/analogs & derivatives , Homocysteine/metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Quaternary , Protein Structure, Secondary , Selenomethionine/metabolism , Sequence Alignment , Zinc/metabolism
5.
Nature ; 413(6857): 752-5, 2001 Oct 18.
Article in English | MEDLINE | ID: mdl-11607036

ABSTRACT

In humans, uracil appears in DNA at the rate of several hundred bases per cell each day as a result of misincorporation of deoxyuridine (dU) or deamination of cytosine. Four enzymes that catalyse the hydrolysis of the glycosylic bond of dU in DNA to yield an apyridiminic site as the first step in base excision repair have been identified in the human genome. The most efficient and well characterized of these uracil-DNA glycosylases is UDG (also known as UNG and present in almost all known organisms), which excises U from single- or double-stranded DNA and is associated with DNA replication forks. We used a hybrid quantum-mechanical/molecular-mechanical (QM/MM) approach to determine the mechanism of catalysis by UDG. In contrast to the concerted associative mechanism proposed initially, we show here that the reaction proceeds in a stepwise dissociative manner. Cleavage of the glycosylic bond yields an intermediate comprising an oxocarbenium cation and a uracilate anion. Subsequent attack by a water molecule and transfer of a proton to D145 result in the products. Surprisingly, the primary contribution to lowering the activation energy comes from the substrate, rather than from the enzyme. This 'autocatalysis' derives from the burial and positioning of four phosphate groups that stabilize the rate-determining transition state. The importance of these phosphates explains the residual activity observed for mutants that lack key residues. A corresponding catalytic mechanism could apply to the DNA glycosylases TDG and SMUG1, which belong to the same structural superfamily as UDG.


Subject(s)
DNA Glycosylases , N-Glycosyl Hydrolases/metabolism , Catalysis , DNA/metabolism , Humans , Models, Molecular , Quantum Theory , Substrate Specificity , Uracil-DNA Glycosidase
6.
Biochem J ; 357(Pt 2): 399-405, 2001 Jul 15.
Article in English | MEDLINE | ID: mdl-11439089

ABSTRACT

Site-directed mutagenesis has been used to characterize the functions of key amino acid residues in the catalytic site of the 'nudix' hydrolase, (asymmetrical) diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) hydrolase (EC 3.6.1.17) from Lupinus angustifolius, the three-dimensional solution structure of which has recently been solved. Residues within the nudix motif, Gly-(Xaa)5-Glu-(Xaa)7-Arg-Glu-Uaa-Xaa-(Glu)2-Xaa-Gly (where Xaa represents unspecified amino acids and Uaa represents the bulky aliphatic amino acids Ile, Leu or Val) conserved in 'nudix enzymes', and residues important for catalysis from elsewhere in the molecule, were mutated and the expressed proteins characterized. The results reveal a high degree of functional conservation between lupin asymmetric Ap4A hydrolase and the 8-oxo-dGTP hydrolase from Escherichia coli. Charged residues in positions equivalent to those that ligate an enzyme-bound metal ion in the E. coli 8-oxo-dGTP hydrolase [Harris, Wu, Massiah and Mildvan (2000) Biochemistry 39, 1655-1674] were shown to contribute to catalysis to similar extents in the lupin enzyme. Mutations E55Q, E59Q and E125Q all reduced kcat markedly, whereas mutations R54Q, E58Q and E122Q had smaller effects. None of the mutations produced a substantial change in the Km)for Ap4A, but several extensively modified the pH-dependence and fluoride-sensitivities of the hydrolase. It was concluded that the precisely positioned glutamate residues Glu-55, Glu-59 and Glu-125 are conserved as functionally significant components of the hydrolytic mechanism in both of these members of the nudix family of hydrolases.


Subject(s)
Acid Anhydride Hydrolases/chemistry , Acid Anhydride Hydrolases/metabolism , Escherichia coli Proteins , Fabaceae/enzymology , Plants, Medicinal , Amino Acid Sequence , Amino Acid Substitution , Bacterial Proteins/chemistry , Binding Sites , Catalysis , Circular Dichroism , Consensus Sequence , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphoric Monoester Hydrolases/chemistry , Protein Structure, Secondary , Pyrophosphatases , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
7.
J Cardiovasc Pharmacol ; 37(5): 571-84, 2001 May.
Article in English | MEDLINE | ID: mdl-11336108

ABSTRACT

Platelet activation in heart disease is important owing to the effects of platelet-derived compounds on myocardial perfusion and cardiac electrophysiology. Diadenosine polyphosphates are secreted from platelets and present in the myocardium, but their electrophysiologic and vasomotor effects are incompletely understood. We used isolated guinea-pig hearts to study the effects of diadenosine triphosphate (Ap3A), tetraphosphate (Ap4A), pentaphosphate (Ap5A), and hexaphosphate (Ap6A) (10 pM-0.1 mM), comparing their actions to those of adenosine, adenosine triphosphate, and non-hydrolyzable Ap4A and Ap5A analogs. Diadenosine polyphosphates (0.1 nM-0.1 microM) transiently reduced coronary perfusion pressure, which recovered during the continued presence of the compounds. At concentrations greater than 0.1 microM effects were maximal and sustained (perfusion pressure decreased from 36.5+/-3.4 to 18.6+/-2.5 mm Hg, p < 0.001, with 1 microM Ap4A). The changes in action potential duration and refractory period developed slowly but were maintained (0.1 nM-1 microM). With 1 nM Ap4A, action potential duration increased from 170.6+/-2.6 to 187.3+/-3.8 ms, p < 0.05, and refractory period increased from 138.5+/-1.6 to 147.9+/-2.0 ms, p < 0.05. Ap4A and its analog reduced QRS duration (from 24.7+/-1.1 to 13.9+/-1.6 ms with 1 microM Ap4A, p < 0.05). P2-purinergic (adenosine triphosphate) receptor antagonism (suramin) reduced perfusion pressure but was without electrophysiologic effect. Other changes in coronary perfusion pressure and electrophysiologic variables associated with Ap4A were not seen in the presence of suramin. P1-(adenosine) antagonism (8-[p-sulfophenyl]theophylline) attenuated the electrophysiologic effects only. Diadenosine polyphosphates have potent cardiac electrophysiologic and coronary vasomotor effects via purinergic receptors, suggesting an important role during platelet activation in acute coronary syndromes.


Subject(s)
Action Potentials/drug effects , Dinucleoside Phosphates/pharmacology , Heart/drug effects , Vasoconstrictor Agents/pharmacology , Vasomotor System/drug effects , Action Potentials/physiology , Animals , Coronary Vessels/drug effects , Coronary Vessels/physiology , Dinucleoside Phosphates/chemistry , Guinea Pigs , Heart/physiology , Heart Rate/drug effects , Heart Rate/physiology , Hydrolysis , Male , Purinergic P1 Receptor Antagonists , Purinergic P2 Receptor Antagonists , Receptors, Purinergic P1/physiology , Receptors, Purinergic P2/physiology , Structure-Activity Relationship , Vasoconstrictor Agents/chemistry , Vasomotor System/physiology
8.
Proc Natl Acad Sci U S A ; 97(10): 5083-8, 2000 May 09.
Article in English | MEDLINE | ID: mdl-10805771

ABSTRACT

Enzymatic transformations of macromolecular substrates such as DNA repair enzyme/DNA transformations are commonly interpreted primarily by active-site functional-group chemistry that ignores their extensive interfaces. Yet human uracil-DNA glycosylase (UDG), an archetypical enzyme that initiates DNA base-excision repair, efficiently excises the damaged base uracil resulting from cytosine deamination even when active-site functional groups are deleted by mutagenesis. The 1.8-A resolution substrate analogue and 2.0-A resolution cleaved product cocrystal structures of UDG bound to double-stranded DNA suggest enzyme-DNA substrate-binding energy from the macromolecular interface is funneled into catalytic power at the active site. The architecturally stabilized closing of UDG enforces distortions of the uracil and deoxyribose in the flipped-out nucleotide substrate that are relieved by glycosylic bond cleavage in the product complex. This experimentally defined substrate stereochemistry implies the enzyme alters the orientation of three orthogonal electron orbitals to favor electron transpositions for glycosylic bond cleavage. By revealing the coupling of this anomeric effect to a delocalization of the glycosylic bond electrons into the uracil aromatic system, this structurally implicated mechanism resolves apparent paradoxes concerning the transpositions of electrons among orthogonal orbitals and the retention of catalytic efficiency despite mutational removal of active-site functional groups. These UDG/DNA structures and their implied dissociative excision chemistry suggest biology favors a chemistry for base-excision repair initiation that optimizes pathway coordination by product binding to avoid the release of cytotoxic and mutagenic intermediates. Similar excision chemistry may apply to other biological reaction pathways requiring the coordination of complex multistep chemical transformations.


Subject(s)
DNA Glycosylases , DNA/chemistry , DNA/metabolism , N-Glycosyl Hydrolases/chemistry , N-Glycosyl Hydrolases/metabolism , Amino Acid Sequence , Binding Sites , Catalysis , Crystallography, X-Ray , DNA Repair , Humans , Mitochondria/enzymology , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Conformation , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Uracil-DNA Glycosidase
9.
Xenobiotica ; 30(3): 219-33, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10752638

ABSTRACT

1. Debrisoquine, a prototypic probe substrate for human cytochrome P4502D6 (CYP2D6), is hydroxylated at the alicyclic C4-position by this enzyme. Phenolic metabolites of debrisoquine (5-, 6-, 7- and 8-hydroxydebrisoquine) have also been reported as in vivo metabolites, but the role of CYP2D6 in their formation is unclear. 2. As part of studies to develop a predictive model of the active site of CYP2D6 using pharmacophore and homology modelling techniques, it became important to determine the precise regioselective hydroxylation of debrisoquine by CYP2D6. 3. Data from studies with human liver microsomes and yeast microsomes containing cDNA-derived CYP2D6 demonstrated unequivocally that debrisoquine was hydroxylated by CYP2D6 at each aromatic site in the molecule, as well as at the alicyclic 4-position. The four phenolic metabolites amounted to > 60% of the total identified products and the pattern of regioselective hydroxylation (4-HD > 7-HD > 6-HD > 8-HD > 5-HD) was similar in both in vitro systems. 4. A pharmacophore model for CYP2D6 indicated that while the hydroxylation of debrisoquine at alternative positions could arise from the substrate adopting multiple binding orientations, the energy constraints for the aromatic hydroxylations were unfavourable. An alternative proposal involving essentially a single binding orientation and a mechanism of hydroxylation based on benzylic radical spin delocalization could satisfactorily rationalize all the hydroxylations of debrisoquine. 5. This latter proposal demonstrates the need to consider the mechanism of oxidation as well as the spatial orientation of the substrate in the development of a predictive model of the active site of CYP2D6.


Subject(s)
Cytochrome P-450 CYP2D6/pharmacology , Debrisoquin/chemistry , Debrisoquin/metabolism , Binding Sites , Chromatography, High Pressure Liquid , Cytochrome P-450 CYP2D6/genetics , DNA, Complementary/metabolism , Humans , Hydrogen Bonding , Hydroxylation , Kinetics , Microsomes, Liver/metabolism , Models, Chemical , Yeasts/metabolism
10.
J Mol Biol ; 302(5): 1165-77, 2000 Oct 06.
Article in English | MEDLINE | ID: mdl-11183782

ABSTRACT

The solution structure of diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L., an enzyme of the Nudix family, has been determined by heteronuclear NMR, using a torsion angle dynamics/simulated annealing protocol based on approximately 12 interresidue NOEs per residue. The structure represents the first Ap4A hydrolase to be determined, and sequence homology suggests that other members will have the same fold. The family of structures shows a well-defined fold comprised of a central four-stranded mixed beta-sheet, a two-stranded antiparallel beta-sheet and three helices (alphaI, alphaIII, alphaIV). The root-mean-squared deviation for the backbone (C',O,N,Calpha) of the rigid parts (residues 9 to 75, 97 to 115, 125 to 160) of the protein is 0.32 A. Several regions, however, show lower definition, particularly an isolated helix (alphaII) that connects two strands of the central sheet. This poor definition is mainly due to a lack of long-range NOEs between alphaII and other parts of the protein. Mapping conserved residues outside of the Nudix signature and those sensitive to an Ap4A analogue suggests that the adenosine-ribose moiety of the substrate binds into a large cleft above the four-stranded beta-sheet. Four conserved glutamate residues (Glu55, Glu58, Glu59 and Glu125) form a cluster that most likely ligates an essential magnesium ion, however, Gly41 also an expected magnesium ligand, is distant from this cluster.


Subject(s)
Acid Anhydride Hydrolases/chemistry , Magnoliopsida/enzymology , Pyrophosphatases/chemistry , Acid Anhydride Hydrolases/antagonists & inhibitors , Acid Anhydride Hydrolases/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Dinucleoside Phosphates/chemistry , Dinucleoside Phosphates/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Ligands , Magnesium/metabolism , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Protein Structure, Tertiary , Pyrophosphatases/antagonists & inhibitors , Pyrophosphatases/metabolism , Sequence Alignment , Solutions , Static Electricity , Nudix Hydrolases
12.
Nucleosides Nucleotides Nucleic Acids ; 19(10-12): 1505-16, 2000.
Article in English | MEDLINE | ID: mdl-11200255

ABSTRACT

2',4'-Dideoxy-4'-methyleneuridine incorporated into oligodeoxynucleotides forms regular B-DNA duplexes as shown by Tm and CD measurements. Such oligomers are not cleaved by the DNA repair enzyme, UDG, which cleaves the glycosylic bond in dU but not in dT nor in dC nucleosides in single stranded and double stranded DNA. Differential binding of oligomers containing carbadU, 4'-thiodU, and dU residues to wild type and mutant UDG proteins identify an essential role for the furanose 4'-oxygen in recognition and cleavage of dU residues in DNA.


Subject(s)
DNA Glycosylases , DNA Repair , N-Glycosyl Hydrolases/metabolism , Nucleotides/metabolism , Base Sequence , Herpesvirus 1, Human/enzymology , Magnetic Resonance Spectroscopy , Surface Plasmon Resonance , Uracil-DNA Glycosidase
13.
J Med Chem ; 41(23): 4439-52, 1998 Nov 05.
Article in English | MEDLINE | ID: mdl-9804684

ABSTRACT

We have synthesized a series of novel analogs of 1, 3-bisphospho-D-glyceric acid, 1,3-BPG,3 and evaluated their binding to phosphoglycerate kinase, PGK (EC 2.7.2.3). Nonscissile methanephosphonic acids replace the two phosphate monoesters of 1, 3-BPG and lead to several stable, tight-binding mimics of this intermediate species in glycolysis. Multiple fluorine substitution for hydrogen in the alpha-methylene groups of the phosphonic acid 1, 3-BPG analogs markedly improves their binding to PGK as determined by NMR analysis. The best ligands bind some 50-100 times more strongly than does the substrate 3-phospho-D-glyceric acid and show a requirement for pKa3 to be generally below 6.0, while the presence of a beta-carbonyl group seems to be of secondary importance.


Subject(s)
Diphosphoglyceric Acids/chemical synthesis , Diphosphonates/chemical synthesis , Phosphoglycerate Kinase/metabolism , Diphosphoglyceric Acids/chemistry , Diphosphoglyceric Acids/metabolism , Diphosphonates/metabolism , Glyceric Acids/metabolism , Hydrogen Bonding , Ligands , Magnetic Resonance Spectroscopy , Protein Binding , Structure-Activity Relationship , Yeasts/enzymology
14.
Nucleosides Nucleotides ; 17(1-3): 301-8, 1998.
Article in English | MEDLINE | ID: mdl-9708352

ABSTRACT

The design and synthesis of analogues of diadenosine 5',5"'-P1,P3-triphosphate that are resistant to pyrophosphate hydrolysis is described in relation to their rôle in signalling and tumorigenesis involving the Fhit protein, the human fragile histidine triad protein, which is a novel Ap3A binding/cleaving protein.


Subject(s)
Dinucleoside Phosphates/chemistry , Neoplasm Proteins , Polyphosphates/chemical synthesis , Proteins/metabolism , Acid Anhydride Hydrolases/metabolism , Binding Sites/physiology , DNA-Binding Proteins/metabolism , Humans , Hydrolysis , Molecular Structure , Stereoisomerism
15.
J Mol Biol ; 279(5): 1137-48, 1998 Jun 26.
Article in English | MEDLINE | ID: mdl-9642090

ABSTRACT

The glycolytic enzyme phosphoglycerate kinase (PGK) catalyzes phosphoryl transfer between 1,3-bis-phosphoglycerate and ADP to form 3-phosphoglycerate and ATP. During catalysis, a major hinge bending motion occurs which brings the N and C-terminal enzyme domains and their bound substrates together and in-line for phosphoryl transfer. We have crystallized Trypanosoma brucei PGK in the presence of the bisubstrate analog, adenylyl 1,1,5,5-tetrafluoropentane-1, 5-bisphosphonate, and solved the structure of this complex in two different crystal forms at 1.6 and 2.0 A resolution, obtained from PEG 8000 and ammonium phosphate solutions, respectively. These high resolution structures of PGK:inhibitor complexes are of particular interest for drug design since Trypanosoma brucei, the causative agent of African sleeping sickness, relies on glycolysis as its sole energy source. In both structures, the inhibitor is bound in a fully extended conformation with its adenosine moiety assuming exactly the same position as in ADP:PGK complexes and with its 5' phosphonate group occupying part of the 1,3-bis-phosphoglycerate binding site. The bisubstrate analog forces PGK to assume a novel, "inhibited" conformation, intermediate in hinge angle between the native structures of open and closed form PGK. These structures of enzyme-inhibitor complexes demonstrate that PGK has two distinct hinge points that can each be independently activated. In the "PEG" structure, the C-terminal hinge is partially activated while the N-terminal hinge point remains in an open state. In the "phosphate" structure, closure of the N-terminal hinge point is also evident. Finally and most unexpectedly, both complex structures also contain a 3 A shift of a helix that lies outside the flexible hinge region. We propose that a transient shift of this helix is a required element of PGK hinge closure during catalysis.


Subject(s)
Models, Molecular , Phosphoglycerate Kinase/chemistry , Protozoan Proteins/chemistry , Trypanosoma brucei brucei/enzymology , Animals , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , Phosphoglycerate Kinase/antagonists & inhibitors , Protein Conformation , Protozoan Proteins/antagonists & inhibitors , Substrate Specificity
16.
Proc Natl Acad Sci U S A ; 95(10): 5484-9, 1998 May 12.
Article in English | MEDLINE | ID: mdl-9576908

ABSTRACT

Alterations in the FHIT gene at 3p14.2 occur as early and frequent events in the development of several common human cancers. The ability of human Fhit-negative cells to form tumors in nude mice is suppressed by stable reexpression of Fhit protein. Fhit protein is a diadenosine P1,P3-triphosphate (ApppA) hydrolase whose fungal and animal homologs form a branch of the histidine triad (HIT) superfamily of nucleotide-binding proteins. Because the His-96 --> Asn substitution of Fhit, which retards ApppA hydrolase activity by seven orders of magnitude, did not block tumor-suppressor activity in vivo, we determined whether this mutation affected ApppA binding or particular steps in the ApppA catalytic cycle. Evidence is presented that His-96 --> Asn protein binds ApppA well and forms an enzyme-AMP intermediate extremely poorly, suggesting that Fhit-substrate complexes are the likely signaling form of the enzyme. The cocrystal structure of Fhit bound to Ado-p-CH2-p-ps-Ado (IB2), a nonhydrolyzable ApppA analog, was refined to 3.1 A, and the structure of His-96 --> Asn Fhit with IB2 was refined to 2.6 A, revealing that two ApppA molecules bind per Fhit dimer; identifying two additional adenosine-binding sites on the dimer surface; and illustrating that His-98 is positioned to donate a hydrogen bond to the scissile bridging oxygen of ApppA substrates. The form of Fhit bound to two ApppA substrates would present to the cell a dramatically phosphorylated surface, prominently displaying six phosphate groups and two adenosine moieties in place of a deep cavity lined with histidines, arginines, and glutamines.


Subject(s)
Acid Anhydride Hydrolases , Neoplasm Proteins , Proteins/chemistry , Animals , Crystallography, X-Ray , Dimerization , Dinucleoside Phosphates/metabolism , Humans , Ligands , Mice , Models, Molecular , Molecular Sequence Data , Protein Conformation , Proteins/genetics , Proteins/metabolism , Static Electricity
17.
Bioorg Med Chem Lett ; 8(5): 515-20, 1998 Mar 03.
Article in English | MEDLINE | ID: mdl-9871609

ABSTRACT

A series of novel, conformationally-restrained bisphosphonate analogues of 1,3-bisphosphoglyceric acid 1 have been synthesised and evaluated as inhibitors of 3-PGK. They are competitive inhibitors of the human enzyme and, especially for certain alpha-halophosphonic acid analogues, both Ki and IC50 values extend into the submicromolar range.


Subject(s)
Diphosphonates/pharmacology , Enzyme Inhibitors/pharmacology , Phosphoglycerate Kinase/antagonists & inhibitors , Diphosphonates/chemistry , Enzyme Inhibitors/chemistry , Humans , Kinetics
18.
Bioorg Med Chem Lett ; 8(18): 2603-8, 1998 Sep 22.
Article in English | MEDLINE | ID: mdl-9873589

ABSTRACT

Stable bisubstrate ligands of phosphoglycerate kinase (PGK) have been synthesized with AMP or ADP conjugated to hydrolytically-stable, symmetrical analogues of 1,3-bisphosphoglycerate and their binding to yeast PGK evaluated. Their Kds decrease with net negative charge, with a penta-anionic analogue 7 showing highest affinity-in accordance with its approximation to the transition state for the reaction catalysed by PGK.


Subject(s)
Adenosine Diphosphate/analogs & derivatives , Adenosine Monophosphate/analogs & derivatives , Phosphoglycerate Kinase/metabolism , Catalysis , Chromatography, High Pressure Liquid , Diphosphoglyceric Acids/chemistry , Diphosphoglyceric Acids/metabolism , Drug Design , Kinetics , Ligands , Magnetic Resonance Spectroscopy , Models, Chemical
19.
J Bone Miner Res ; 12(9): 1358-67, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9286751

ABSTRACT

Clodronate, alendronate, and other bisphosphonates are widely used in the treatment of bone diseases characterized by excessive osteoclastic bone resorption. The exact mechanisms of action of bisphosphonates have not been identified but may involve a toxic effect on mature osteoclasts due to the induction of apoptosis. Clodronate encapsulated in liposomes is also toxic to macrophages in vivo and may therefore be of use in the treatment of inflammatory diseases. It is generally believed that bisphosphonates are not metabolized. However, we have found that mammalian cells in vitro (murine J774 macrophage-like cells and human MG63 osteosarcoma cells) can metabolize clodronate (dichloromethylenebisphosphonate) to a nonhydrolyzable adenosine triphosphate (ATP) analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, which could be detected in cell extracts by using fast protein liquid chromatography. J774 cells could also metabolize liposome-encapsulated clodronate to the same ATP analog. Liposome-encapsulated adenosine 5'-(beta, gamma-dichloromethylene) triphosphate was more potent than liposome-encapsulated clodronate at reducing the viability of cultures of J774 cells and caused both necrotic and apoptotic cell death. Neither alendronate nor liposome-encapsulated alendronate were metabolized. These results demonstrate that the toxic effect of clodronate on J774 macrophages, and probably on osteoclasts, is due to the metabolism of clodronate to a nonhydrolyzable ATP analog. Alendronate appears to act by a different mechanism.


Subject(s)
Adenosine Triphosphate/analogs & derivatives , Analgesics, Non-Narcotic/metabolism , Clodronic Acid/metabolism , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/toxicity , Alendronate/metabolism , Alendronate/pharmacology , Analgesics, Non-Narcotic/administration & dosage , Analgesics, Non-Narcotic/pharmacology , Apoptosis/drug effects , Cell Survival/drug effects , Cells, Cultured , Clodronic Acid/administration & dosage , Clodronic Acid/pharmacology , Diphosphonates/pharmacology , Humans , Liposomes , Microscopy, Fluorescence
20.
Eur J Pharmacol ; 332(1): 35-42, 1997 Jul 30.
Article in English | MEDLINE | ID: mdl-9298923

ABSTRACT

Findings in peripheral tissues that diadenosine polyphosphates (Ap(n)As) activate 5'-nucleotidase activity and inhibit adenosine kinase activity in vitro led us to test the hypothesis that Ap(n)As and analogues thereof, through such actions on purine enzymes, increase brain levels of endogenous adenosine in vivo. Accordingly, we tested Ap(n)As for their effects on the in vitro activities of adenosine kinase, adenosine deaminase, AMP deaminase and 5'-nucleotidase and, following unilateral microinjections in rat striatum, on in vivo levels of endogenous adenosine. Adenosine kinase activity was not affected significantly by 5',5'''-P1,P2-diadenosine pyrophosphate (Ap2A) or by 5',5'''-P1,P3-diadenosine triphosphate (Ap3A), but was inhibited by 5',5'''-P1,P4-diadenosine tetraphosphate (Ap4A), 5',5'''-P1,P5-diadenosine pentaphosphate (Ap5A) and 5',5'''-P1,P6-diadenosine hexaphosphate (Ap6A); apparent IC50 values were 5.0, 3.3 and 500 microM, respectively. Inhibition of adenosine kinase activity by Ap4A and the four metabolically stable analogues of Ap4A tested was uncompetitive. Following unilateral intrastriatal injections, adenosine levels, relative to uninjected contralateral striatum, were decreased significantly (P < 0.05) by 48% with Ap4A and by 37% with AppCH2ppA, a metabolically stable analogue of Ap4A. Striatal levels of adenosine were not affected significantly by Ap5A or Ap6A. Cytosolic, but not particulate 5'-nucleotidase activity was inhibited and AMP deaminase activity was increased by some Ap(n)As. Although adenosine kinase inhibitors increase levels of endogenous adenosine and we showed here that Ap(n)As were potent inhibitors of this enzyme, these particular actions of Ap(n)As were not consistent with their effects on levels of endogenous adenosine.


Subject(s)
Adenosine Kinase/antagonists & inhibitors , Adenosine/metabolism , Anti-Arrhythmia Agents/metabolism , Brain Chemistry/drug effects , Dinucleoside Phosphates/pharmacology , Vasoconstrictor Agents/pharmacology , 5'-Nucleotidase/drug effects , AMP Deaminase/drug effects , Adenosine Kinase/metabolism , Animals , Dose-Response Relationship, Drug , Male , Rats , Rats, Sprague-Dawley
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