A series of 2-fluoro-4-alkene and 2-fluoro-4-alkyne substrate analogues were synthesized and examined as potential inhibitors of three enzymes: 4-oxalocrotonate tautomerase (4-OT) and vinylpyruvate ...hydratase (VPH) from the catechol meta-fission pathway and a closely related 4-OT homologue found in Bacillus subtilis designated YwhB. All of the compounds were potent competitive inhibitors of 4-OT with the monocarboxylated 2E-fluoro-2,4-pentadienoate and the dicarboxylated 2E-fluoro-2-en-4-ynoate being the most potent. Despite the close mechanistic and structural similarities between 4-OT and YwhB, these compounds were significantly less potent inhibitors of YwhB with K i values ranging from 5- to 633-fold lower than those determined for 4-OT. The study of VPH is complicated by the fact that the enzyme is only active as a complex with the metal-dependent 4-oxalocrotonate decarboxylase (4-OD), the enzyme following 4-OT in the catechol meta-fission pathway. A structure-based sequence analysis identified 4-OD as a member of the fumarylacetoacetate hydrolase (FAH) superfamily and implicated Glu-109 and Glu-111 as potential metal-binding ligands. Changing these residues to a glutamine verified their importance for enzymatic activity and enabled the production of soluble E109Q4-OD/VPH or E111Q4-OD/VPH complexes, which retained full hydratase activity but had little decarboxylase activity. Subsequent incubation of the E109Q4-OD/VPH complex with the substrate analogues identified the 2E and 2Z isomers of the monocarboxylated 2-fluoropent-2-en-4-ynoate as competitive inhibitors. The combined results set the stage for crystallographic studies of 4-OT, YwhB, and VPH using these inhibitors as ligands.
The compound, 2-oxo-3-pentynoate, has been synthesized and tested as an inhibitor of the enzyme 4-oxalocrotonate tautomerase. The enzyme is rapidly and irreversibly inactivated by the acetylenic ...product analogue in a time-dependent fashion. The enzyme displays saturation kinetics and is protected from inactivation by the presence of substrate. These observations are consistent with inactivation taking place at the active site. Partial reactivation (∼18%) occurs by incubating the inactivated enzyme with 10 mM hydroxylamine (pH 7.3). The partition ratio, determined to be ∼0.4, suggests that the inactivation of 4-OT by 2-oxo-3-pentynoate shows half-of-the-sites stoichiometry. The same phenomenon is observed in the inactivation of 4-OT by 3-bromopyruvate and can be explained by examination of the crystal structure. Mass spectral analysis shows that a single residue is modified on the enzyme which has been localized to the nine residue amino-terminal fragment Pro-1 to Glu-9. It can be reasonably concluded that Pro-1 is the site of covalent attachment. Inactivation of 4-OT can occur by either a Michael addition of 4-OT to C-4 of 2-oxo-3-pentynoate or by the enzyme-catalyzed rearrangement of 2-oxo-3-pentynoate to an allene derivative which alkylates Pro-1. These results provide the foundation for the use of 2-oxo-3-pentynoate in future mechanistic studies and as a ligand in an inactivated 4-OT complex that can be studied by X-ray crystallography. Finally, 2-oxo-3-pentynoate is an acetylene analogue of a variety of 2-oxo acids and as such may have general utility as an inhibitor of reactions that bind and process these compounds.
Macrophage migration inhibitory factor (MIF) is an important immunoregulatory protein that has been implicated in several inflammatory diseases. MIF also has a phenylpyruvate tautomerase (PPT) ...activity, the role of which remains elusive in these biological activities. The acetylene compound, 2-oxo-4-phenyl-3-butynoate (2-OPB), has been synthesized and tested as a potential irreversible inhibitor of its enzymatic activity. Incubation of the compound with MIF results in the rapid and irreversible loss of the PPT activity. Mass spectral analysis established that the amino-terminal proline, previously implicated as a catalytic base in the PPT-catalyzed reaction, is the site of covalent modification. Inactivation of the PPT activity likely occurs by a Michael addition of Pro-1 to C-4 of the inhibitor. Attempts to crystallize the inactivated complex to confirm the structure of the adduct on the covalently modified Pro-1 by X-ray crystallography were not successful. Nor was it possible to unambiguously interpret electron density observed in the active sites of the native crystals soaked with the inhibitor. This may be due to crystal packing in that the side chain of Glu-16 from an adjacent trimer occupies one active site. However, this crystal contact may be partially responsible for the high-resolution quality of these MIF crystals. Nonetheless, because MIF is a member of the tautomerase superfamily, a group of structurally homologous proteins that share a β–α–β structural motif and a catalytic Pro-1, 2-OPB may find general use as a probe of tautomerase superfamily members that function as PPTs.
The amino-terminal proline of 4-oxalocrotonate tautomerase (4-OT) functions as the general base catalyst in the enzyme-catalyzed isomerization of β,γ-unsaturated enones to their α,β-isomers because ...of its unusually low pK a of 6.4 ± 0.2, which is 3 units lower than that of the model compound, proline amide. Recent studies show that this abnormally low pK a is not due to the electrostatic effects of nearby cationic residues (Arg-11, Arg-39, and Arg-61) Czerwinski, R. M., Harris, T. K., Johnson, Jr., W. H., Legler, P. M., Stivers, J. T., Mildvan, A. S., and Whitman, C. P. (1999) Biochemistry 38, 12358−12366. Hence, it may result solely from a low local dielectric constant of 14.7 ± 0.8 at the otherwise hydrophobic active site. Support for this mechanism comes from the study of mutants of the active site Phe-50, which is 5.8 Å from Pro-1 and is one of 12 apolar residues within 9 Å of Pro-1. Replacing Phe-50 with Tyr does not significantly alter k cat or K m and results in a pK a of 6.0 ± 0.1 for Pro-1 as determined by 15N NMR spectroscopy, comparable to that observed for wild type. 1H-15N HSQC and 3D 1H-15N NOESY HSQC spectra of the F50Y mutant demonstrate its conformation to be very similar to that of the wild-type enzyme. In the F50Y mutant, the pK a of Tyr-50 is increased by two units from that of a model compound N-acetyl-tyrosine amide to 12.2 ± 0.3, as determined by UV and 1H NMR titrations, yielding a local dielectric constant of 13.4 ± 1.7, in agreement with the value of 13.7 ± 0.3 determined from the decreased pK a of Pro-1 in this mutant. In the F50A mutant, the pK a of Pro-1 is 7.3 ± 0.1 by 15N NMR titration, comparable to the pK a of 7.6 ± 0.2 found in the pH vs k cat/K m rate profile, and is one unit greater than that of the wild-type enzyme, indicating an increase in the local dielectric constant to a value of 21.2 ± 2.6. A loss of structure of the β-hairpin from residues 50 to 57, which covers the active site, and is the site of the mutation, is indicated by the disappearance in the F50A mutant of four interstrand NOEs and one turn NOE found in wild-type 4-OT. 1H-15N HSQC spectra of the F50A mutant reveal widespread and large changes in the backbone 15N and NH chemical shifts including those of Gly residues 48, 51, 53, and 54 causing their loss of dispersion at 23 °C and their disappearance at 43 °C due to rapid exchange with solvent. These observations confirm that the active site of the F50A mutant is more accessible to the external aqueous environment, causing an increase in the local dielectric constant and in the pK a of Pro-1. In addition, the F50A mutation decreased k cat 167-fold and increased K m 11-fold from those of the wild-type enzyme, suggesting an important role for the hydrophobic environment in catalysis, beyond that of decreasing the pK a of Pro-1. The F50I and F50V mutations destabilize the protein and decrease k cat by factors of 58 and 1.6, and increase K m by 3.3- and 3.8-fold, respectively.
Carbamoyl phosphate synthetase from Escherichia coli catalyzes the synthesis of carbamoyl phosphate from bicarbonate, ammonia, and two molecules of MgATP. The enzyme is composed of two nonidentical ...subunits. The small subunit catalyzes the hydrolysis of glutamine to glutamate and ammonia. The large subunit catalyzes the formation of carbamoyl phosphate and has the binding sites for bicarbonate, ammonia, MgATP, and the allosteric ligands IMP, UMP, and ornithine. The allosteric ligands are believed to bind to the extreme C-terminal portion of the large subunit. Truncation mutants were constructed to investigate the allosteric binding domain. Stop codons were introduced at various locations along the carB gene in order to delete amino acids from the carboxy-terminal end of the large subunit. Removal of 14-119 amino acids from the carboxy-terminal end of the large subunit resulted in significant decreases in all of the enzymatic activities catalyzed by the enzyme. A 40-fold decrease in the glutamine-dependent ATPase activity was observed for the delta 14 truncation. Similar losses in activity were also observed for the delta 50, delta 65, delta 91, and delta 119 mutant proteins. However, formation of carbamoyl phosphate was detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects were observed for UMP with either the delta 91 or delta 119 truncation mutants, but alterations in the catalytic activity were observed in the presence of ornithine even after the removal of the last 119 amino acids from the large subunit of CPS. Six conserved amino acids within the allosteric domain were mutated.
Macrophage migration inhibitory factor (MIF), an immunoregulatory protein, exhibits a phenylpyruvate tautomerase (PPT) activity. The catalytic mechanism of this activity has recently attracted ...attention in an effort to determine whether there is a relationship between the PPT activity and the role of MIF in various immune and inflammatory processes. One of the active site residues is lysine-32, which is postulated to play two roles: it assists in substrate binding through an interaction with a carboxylate oxygen at C-1 of phenylpyruvate, and it may be partially responsible for lowering the pK a of the catalytic base, Pro-1. The role of Lys-32 has been investigated by changing it to an alanine and an arginine and determining the kinetic parameters, the stereoselectivity, the competitive inhibition, and the pH dependence of the resulting K32A- and K32R-catalyzed reactions. For the K32R mutant, these properties are mostly comparable to those determined for the wild type with two exceptions. There is a modest decrease in the stereoselectivity of the reaction and in the binding affinity of the competitive inhibitor, (E)-2-fluoro-p-hydroxycinnamate. These differences are likely due to the increased steric bulk of arginine. For the K32A mutant, there are 11- and 12-fold decreases in k cat and k cat/K m, respectively, using phenylenolpyruvate. Part of the decrease in activity can be attributed to the observed increase of 1.3 units in the pK a of Pro-1. It was also found that the loss of the electrostatic interaction did not significantly affect the stereoselectivity of the K32A-catalyzed reaction, although it did result in a decrease in the binding affinity of the competitive inhibitor. The combination of these results indicates that the primary function of Lys-32 in the PPT activity of MIF is to lower the pK a of Pro-1. The interactions responsible for the stereoselectivity of the PPT activity were further delineated by examining the wild type- and K32A-catalyzed reactions with an alternate substrate, 2-hydroxy-2,4-pentadienoate, in which the phenyl group of phenylenolpyruvate is replaced with a double bond. The effect of this substitution is moderate as evidenced by the observation that the ketonization of 2-hydroxy-2,4-pentadienoate by the wild type protein is more stereoselective than the K32R-catalyzed ketonization of phenylenolpyruvate but not as stereoselective as the K32A-catalyzed ketonization of phenylenolpyruvate. However, the low degree of stereoselectivity observed for the K32A-catalyzed reaction indicates that an electrostatic interaction between the protein and 2-hydroxy-2,4-pentadienoate is now crucial.
The speciation of U and Pu in soil and concrete from Rocky Flats and in particles from soils from Chernobyl, Hanford, Los Alamos, and McGuire Air Force Base and bottom sediments from Mayak was ...determined by a combination of X-ray absorption fine structure (XAFS) spectroscopy and X-ray fluorescence (XRF) element maps. These experiments identify four types of speciation that sometimes may and other times do not exhibit an association with the source terms and histories of these samples: relatively well ordered PuO2+x and UO2+x that had equilibrated with O2 and H2O under both ambient conditions and in fires or explosions; instances of small, isolated particles of U as UO2+x , U3O8, and U(VI) species coexisting in close proximity after decades in the environment; alteration phases of uranyl with other elements including ones that would not have come from soils; and mononuclear Pu–O species and novel PuO2+x -type compounds incorporating additional elements that may have occurred because the Pu was exposed to extreme chemical conditions such as acidic solutions released directly into soil or concrete. Our results therefore directly demonstrate instances of novel complexity in the Å and μm-scale chemical speciation and reactivity of U and Pu in their initial formation and after environmental exposure as well as occasions of unexpected behavior in the reaction pathways over short geological but significant sociological times. They also show that incorporating the actual disposal and site conditions and resultant novel materials such as those reported here may be necessary to develop the most accurate predictive models for Pu and U in the environment.
cDNA encoding DNA topoisomerase I from
Physarum polycephalum was isolated from a poly(A)
+-primed library (3′-region) and by PCR (5′-region). The coding region of cDNA was 3045
bp, encoding a ...polypeptide of molecular mass of 112
kDa. Identity between predicted amino acids sequences of conserved domains and corresponding domains from another eukaryotic type I DNA topoisomerases varied from 33.2 to 53.5% for the core domain and from 33.8 to 57.4% for the C-terminal domain. A peculiar feature of
Physarum DNA topoisomerase I was a stretch of repeated KPAX…X motifs in the N-terminal domain of the polypeptide. Although treatment of the plasmodia with db-cAMP increased relaxing activity of the DNA topoisomerase I several-fold, there was only a slight increase in the mRNA level.
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