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C H Williams

Publications and source records attributed to C H Williams.

At least 19 recordsLinked to original sources
PubMed2006

Specific inhibitors of Plasmodium falciparum thioredoxin reductase as potential antimalarial agents.

Plasmodium falciparum thioredoxin reductase (PfTrxR: NADPH+Trx(S)2+H+<-->NADP++Trx(SH)2) is a high Mr flavin-dependent TrxR that reduces thioredoxin (Trx) via a CysXXXXCys pair located penultimately to the C-terminal Gly. In this respect, PfTrxR differs significantly from its human counterpart which bears a Cys-Sec redox pair at the same position. PfTrxR is essentially involved in antioxidant defense and redox regulation of the parasite and has been previously validated by knock-out studies as a potential drug target for malaria chemotherapy. Moreover, human TrxR is present in most cancer cells at levels tenfold higher than in normal cells. Here we report the discovery of a series of potent inhibitors of PfTrxR. The three most promising inhibitors, 3(IC50(PfTrxR)=2 microM and IC50(hTrxR)=50 microM), 7(IC50(PfTrxR)=2 microM and IC50(hTrxR)=140 microM), and 11(IC50(PfTrxR)=0.5 microM and IC50(hTrxR)=4 microM) were selective for the parasite enzyme. Detailed mechanistic characterization of the effects of these compounds on the PfTrxR-catalyzed reaction showed clear uncompetitive inhibition with respect to both substrate and cofactor. For the most specific PfTrxR inhibitor 7, an alkylation mechanism study based on a thiol conjugation model was performed. Furthermore, all three compounds were active in the lower micromolar range on the chloroquine-resistant P. falciparum strain K1 in vitro.

Animals↗
PubMed2001

Disseminated intravascular coagulation in sepsis.

Disseminated intravascular coagulation (DIC) has been considered a rather rare syndrome characterized by severe bleeding. In fact, both of these beliefs are wrong. Bleeding is fairly rare in DIC. The clotting parameters are usually normal unless the DIC is fulminating. It is usually thought that fibrinogen may be low or absent in DIC. However, afibrinogenemia is rare. Fibrinogen is usually high in DIC because of the high rate of fibrinogen manufacture by the liver in response to stress. DIC is very common and most cases are never diagnosed. This is because it has been hard to find fibrin thrombi in autopsy cases and because acute severe bleeding is uncommon. The reason fibrin thrombi are rare may be because they have been lysed by endogenous fibrinolytic enzymes before the autopsy. The appearance of endogenous fibrinolytic response could be a defense mechanism to lyse the microclots of DIC. In fact, this response is often successful. This defense can be aided by the administration of plasminogen activators that will lyse the clots. Heparin has been used for the treatment of DIC but has proved useless and is, in fact, dangerous. This is because heparin will not dissolve clots and may actually promote platelet agglutination. Administration of plasminogen activators will actually prevent bleeding diathesis.

Animals↗
PubMed2001

Exploring the conformational equilibrium of E. coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy.

The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy. The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably. Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of approximately 1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S. Long-lived fluorescence with two time constants in the range of 0.2-1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation. The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation. The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation. Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation. Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation. Specific binding of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model. Raising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S. High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation.

Escherichia coli↗
PubMed2001

Treatment of severe acute respiratory distress syndrome: a final report on a phase I study.

Adult respiratory distress syndrome (ARDS) has a high mortality. Its only effective treatment is respiratory therapy. If this fails mortality is probably 100 per cent. No other treatment for ARDS has proved effective including "magic bullets." Twenty patients suffering from ARDS secondary to trauma and/or sepsis failed to respond to treatment with mechanical ventilation and positive end-expiratory pressure. On the assumption that disseminated intravascular coagulation initiates ARDS by occluding the pulmonary microcirculation with microclots, the patients were treated with plasminogen activators. The patients responded with significant improvement in partial pressure of oxygen in arterial blood. No bleeding occurred and clotting parameters remained normal. We conclude that ARDS can be safely treated with plasminogen activator.

Adolescent↗
PubMed2000

Kinetic characterization of glutathione reductase from the malarial parasite Plasmodium falciparum. Comparison with the human enzyme.

The homodimeric flavoenzyme glutathione reductase (GR) maintains high intracellular concentrations of the antioxidant glutathione (GSSG + NADPH + H(+) <--> 2 GSH + NADP(+)). Due to its central function in cellular redox metabolism, inhibition of GR from the malarial parasite Plasmodium falciparum represents an important approach to antimalarial drug development; therefore, the catalytic mechanism of GR from P. falciparum has been analyzed and compared with the human host enzyme. The reductive half-reaction is similar to the analogous reaction with GR from other species. The oxidative half-reaction is biphasic, reflecting formation and breakdown of a mixed disulfide between the interchange thiol and GSH. The equilibrium between the E(ox)-EH(2) and GSSG-GSH couples has been modeled showing that the Michaelis complex, mixed disulfide-GSH, is the predominant enzyme form as the oxidative half-reaction progresses; rate constants used in modeling allow calculation of an K(eq) from the Haldane relationship, 0.075, very similar to the K(eq) of the same reaction for the yeast enzyme (0.085) (Arscott, L. D., Veine, D. M., and Williams, C. H., Jr. (2000) Biochemistry 39, 4711-4721). Enzyme-monitored turnover indicates that E(FADH(-))(S-S). NADP(+) and E(FAD)(SH)(2).NADPH are dominant enzyme species in turnover. Since the individual forms of the enzyme differ in their susceptibility to inhibitors, the prevailing states of GR in the cell are of practical relevance.

Animals↗
PubMed2000

Twists in catalysis: alternating conformations of Escherichia coli thioredoxin reductase.

In thioredoxin reductase (TrxR) from Escherichia coli, cycles of reduction and reoxidation of the flavin adenine dinucleotide (FAD) cofactor depend on rate-limiting rearrangements of the FAD and NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) domains. We describe the structure of the flavin-reducing conformation of E. coli TrxR at a resolution of 3.0 angstroms. The orientation of the two domains permits reduction of FAD by NADPH and oxidation of the enzyme dithiol by the protein substrate, thioredoxin. The alternate conformation, described by Kuriyan and co-workers, permits internal transfer of reducing equivalents from reduced FAD to the active-site disulfide. Comparison of these structures demonstrates that switching between the two conformations involves a "ball-and-socket" motion in which the pyridine nucleotide-binding domain rotates by 67 degrees.

Binding Sites↗
PubMed2000

Kinetic investigation of the specificity of porcine brain thyrotropin-releasing hormone-degrading ectoenzyme for thyrotropin-releasing hormone-like peptides.

Evidence indicates that neuronally released thyrotropin-releasing hormone (TRH) is selectively inactivated by TRH-degrading ectoenzyme (TRH-DE) (EC ). TRH-DE inhibitors may be used to enhance the therapeutic actions of TRH and to investigate the functions of TRH and TRH-DE in the central nervous system. Although TRH-DE appears to exhibit a high degree of specificity toward TRH, systematic specificity studies, which would facilitate inhibitor design, have not been previously conducted for this enzyme. In this paper we present the first description of TRH-DE specificity across a directed peptide library in which the histidyl (P(1)') residue of TRH was replaced by a series of amino acids. Peptides were synthesized using standard solid phase chemistry. Kinetic parameters were measured either by continuous or discontinuous fluorometric assays or by quantitative high pressure liquid chromatography. The P(1)' residue was found to influence significantly both the ability of the peptides to bind to TRH-DE, as measured by their K(i) values, and the ability of TRH-DE to catalyze their hydrolysis. Moderately bulky, uncharged P(1)' residues were found to bind preferentially to TRH-DE. Results from this screen provide valuable information for the development of TRH-DE inhibitors and have led to the identification of two potent, reversible TRH-DE inhibitors, l-pyroglutamyl-l-asparaginyl-l-prolineamide (K(i) = 17.5 micrometer) and Glp-Asn-Pro-7-amido-4-methyl coumarin (K(i) = 0.97 micrometer).

Aminopeptidases↗
PubMed2000

Mixed disulfide with glutathione as an intermediate in the reaction catalyzed by glutathione reductase from yeast and as a major form of the enzyme in the cell.

Glutathione reductase catalyzes the reduction of glutathione disulfide by NADPH. The FAD of the reductase is reduced by NADPH, and reducing equivalents are passed to a redox-active disulfide to complete the first half-reaction. The nascent dithiol of two-electron reduced enzyme (EH(2)) interchanges with glutathione disulfide forming two molecules of glutathione in the second half-reaction. It has long been assumed that a mixed disulfide (MDS) between one of the nascent thiols and glutathione is an intermediate in this reaction. In addition to the nascent dithiol composed of Cys(45) and Cys(50), the enzyme contains an acid catalyst, His(456), having a pK(a) of 9.2 that protonates the first glutathione (residue numbers refer to the yeast enzyme sequence). Reduction of yeast glutathione reductase by glutathione and reoxidation of EH(2) by glutathione disulfide indicate that the mixed disulfide accumulates, in particular, at low pH. The reaction of glutathione disulfide with EH(2) is stoichiometric in the absence of an excess of glutathione. The equilibrium position among E(ox), MDS, and EH(2) is determined by the glutathione concentration and is not markedly influenced by pH between 6.2 and 8.5. The mixed disulfide is the principal product in the reaction of glutathione with oxidized enzyme (E(ox)) at pH 6. 2. Its spectrum can be distinguished from that of EH(2) by a slightly lower thiolate (Cys(50))-FAD charge-transfer absorbance at 540 nm. The high GSH/GSSG ratio in the cytoplasm dictates that the mixed disulfide will be the major enzyme species.

Catalysis↗
PubMed2000

Thioredoxin reductase two modes of catalysis have evolved.

Thioredoxin reductase (EC 1.6.4.5) is a widely distributed flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin. Thioredoxin plays several key roles in maintaining the redox environment of the cell. Like all members of the enzyme family that includes lipoamide dehydrogenase, glutathione reductase and mercuric reductase, thioredoxin reductase contains a redox active disulfide adjacent to the flavin ring. Evolution has produced two forms of thioredoxin reductase, a protein in prokaryotes, archaea and lower eukaryotes having a Mr of 35 000, and a protein in higher eukaryotes having a Mr of 55 000. Reducing equivalents are transferred from the apolar flavin binding site to the protein substrate by distinct mechanisms in the two forms of thioredoxin reductase. In the low Mr enzyme, interconversion between two conformations occurs twice in each catalytic cycle. After reduction of the disulfide by the flavin, the pyridine nucleotide domain must rotate with respect to the flavin domain in order to expose the nascent dithiol for reaction with thioredoxin; this motion repositions the pyridine ring adjacent to the flavin ring. In the high Mr enzyme, a third redox active group shuttles the reducing equivalent from the apolar active site to the protein surface. This group is a second redox active disulfide in thioredoxin reductase from Plasmodium falciparum and a selenenylsulfide in the mammalian enzyme. P. falciparum is the major causative agent of malaria and it is hoped that the chemical difference between the two high Mr forms may be exploited for drug design.

Animals↗
PubMed2000

Prolactin stimulation of iodide uptake and incorporation into protein is polyamine-dependent in mouse mammary gland explants.

Previous studies have demonstrated that the prolactin stimulation of most lactational processes (casein, lactose, and triglyceride synthesis) requires an earlier stimulating effect of prolactin on the synthesis of the polyamines. Spermidine appears to be the specific polyamine required for prolactin to enhance milk product synthesis. Inorganic iodide is present in milk at more than an order of magnitude higher concentration than that of the maternal plasma. Since prolactin stimulates iodide accumulation in milk, the goal of these studies was to determine the role of the polyamines in this hormone response. Two drugs were employed in these studies: DFMO (difluoromethylornithine), which inhibits ornithine decarboxylase, and MGBG [methylglyoxal bis(guanyl-hydrazone)], which inhibits S-adenosyl methionine decarboxylase. In mammary gland explants from midpregnant (10-14 days of pregnancy) mice, MGBG at 100 microM abolished the prolactin stimulation of iodide uptake and incorporation into milk proteins, whereas DFMO caused a concentration-dependent inhibition of the PRL response. Selected sensitivity of the MGBG and DFMO inhibitions was validated by a reversal of the drug inhibitions with the addition of 1 mM spermidine to the culture medium. These data suggest that the polyamine signaling pathway is involved in the prolactin stimulation of iodide uptake into milk.

Adenosylmethionine Decarboxylase↗
PubMed1999

Inhibition of prolylendopeptidase does not affect gamma-secretase processing of amyloid precursor protein in a human neuroblastoma cell line.

Abeta peptides are major components of the amyloid plaques that characterize Alzheimer's disease. The enzyme activities (beta- and gamma-secretases) involved in generating Abeta from amyloid precursor protein (APP) are unidentified. It has been suggested that prolylendopeptidase (PEP), an oligopeptidase that normally cleaves after proline residues, could also cleave after the alanine at position 42 of Abeta to generate Abeta42. We investigated whether inhibition of PEP activity in human neuroblastoma cells affected Abeta levels in cell culture media. An SH-SY5Y cell line expressing SPA4CT, encoding the C-terminal 100 residues of APP and the signal sequence, was used. Only gamma-secretase activity is required for Abeta production in this cell line. The PEP inhibitor Fmoc-AlaPro-CN (10 microM) reduced PEP activity in these cells by approximately 95% in the absence of significant toxicity, but had no effect on Abeta40 or Abeta42 levels in cell culture media. We conclude that PEP is unlikely to be involved in gamma-secretase processing of APP.

Amino Acids↗
PubMed1999

Development of a continuous, fluorometric coupled enzyme assay for thyrotropin-releasing hormone-degrading ectoenzyme.

Thyrotropin-releasing hormone degrading-ectoenzyme (TRH-DE) (EC 3.4. 19.6), removes the N-terminal pyroglutamyl residue of thyrotropin-releasing hormone (TRH). Discontinuous assays have been used to measure TRH-DE activity; however, a continuous assay is needed to make reliable measurements of initial rates and facilitate kinetic studies. Presented is a continuous, coupled enzyme assay for TRH-DE in which TRH-DE hydrolyzed the substrate, pyroglutamyl-histidyl-prolylamido-4-methyl coumarin (TRHMCA), to give His-ProMCA, which was then cleaved by dipeptidyl peptidase IV (EC 3.4.14.5) to give 7-amino-4-methyl coumarin (MCA). Reaction progress was monitored continuously by measuring the increase in MCA fluorescence. This assay should be especially useful for rapid screening of potential TRH-DE inhibitors. A previously reported discontinuous assay, where nonenzymatic cyclization at 80 degrees C was used to liberate MCA from His-ProMCA, was found to underestimate the amount of product formed. A modified procedure that avoids this is presented. Initial rates and kinetic parameters for TRHMCA hydrolysis by TRH-DE determined using this modified assay correspond with those determined by the continuous assay. Discontinuous and continuous assays gave K(m) values for TRHMCA of 3.4 +/- 0.7 microM (n = 5) and 3.8 +/- 0.5 microM (n = 5), respectively. K(i) values determined by the discontinuous assay for TRH and TRH-OH were 35 +/- 4 microM (n = 3) and 311 +/- 31 microM (n = 5), respectively.

Aminopeptidases↗
PubMed1999

The roles of two amino acid residues in the active site of L-lactate monooxygenase. Mutation of arginine 187 to methionine and histidine 240 to glutamine.

Lactate monooxygenase (LMO) catalyzes the conversion of L-lactate to acetate, CO(2), and water with the incorporation of molecular oxygen. Arginine 187 of LMO is highly conserved within the family of L-alpha-hydroxyacid oxidizing enzymes (Lê, K. H. D., and Lederer, F. (1991) J. Biol. Chem. 266, 20877-20881). By comparison with the equivalent residue in flavocytochrome b(2) from Saccharomyces cerevisiae (Pike, A. D., Chapman, S. K, Manson, F. D. C,. Reid, G. A. , Gondry, M., and Lederer, F. (1996) in Flavins and Flavoproteins (Stevenson, K. J., Massey, V., and Williams, C. H., Jr., eds) pp. 571-574, University of Calgary Press, Calgary, AB, Canada), arginine 187 might be expected to have an important role in catalytic efficiency and substrate binding in LMO. Histidine 240 is predicted to be close to the substrate binding site of LMO, although it is not conserved within the enzyme family. Arginine 187 has been replaced with methionine (R187M), and histidine 240 has been replaced with glutamine (H240Q). L-Lactate oxidation by R187M is very slow. The binding of L-lactate to the mutant enzyme appears to be very weak, as is the binding of oxalate, a transition state analogue. The binding of pyruvate to the reduced enzyme is also very weak, resulting in complete uncoupling of enzyme turnover, with H(2)O(2) and pyruvate as the final products. In addition, anionic forms of the flavin are unstable. The K(d) for sulfite is increased nearly 400-fold by this mutation. The semiquinone form of R187M is also thermodynamically unstable, although the overall midpoint potential for the two-electron reduction of R187M is only 34 mV lower than for the wild-type enzyme. H240Q more closely resembles the wild-type enzyme. The steady-state activity of H240Q is completely coupled. The k(cat) is similar to that for the wild-type enzyme.

Arginine↗
PubMed1999

A continuous fluorometric assay for leukotriene D4 hydrolase.

A fluorogenic substrate for assay of leukotriene D4 hydrolase (LTDase; EC 3.4.13.19) has been prepared and evaluated, using enzyme purified from porcine kidney. The compound is based on internal quenching of the synthetic, fluorescent amino acid d, l-2-amino-3-(7-methoxy-4-coumaryl)propanoic acid (d,l-Amp) by a 2, 4-dinitrophenyl (DNP) group. The compound is epsilon-DNP-l-Lys-d-Amp which incorporates the D-isomer of Amp to exploit the unique ability among mammalian peptidases for LTDase to hydrolyze peptides containing a d-amino acid in the C-terminal position. epsilon-DNP-l-Lys-d-Amp was found to be an excellent substrate for LTDase, with Km value of 370 microoffUnder the conditions of assay, the substrate was without noticeable quenching effect on the fluorescence of the product (d-Amp) liberated by the action of LTDase. Using porcine kidney microvillar membranes, which contain a battery of peptidases, the specific inhibitor of LTDase, cilastatin, completely inhibited the breakdown of epsilon-DNP-l-Lys-d-Amp, indicating that the substrate is selective for LTDase.

Animals↗
PubMed1999

Thioredoxin reductase from Plasmodium falciparum: evidence for interaction between the C-terminal cysteine residues and the active site disulfide-dithiol.

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin by NADPH. TrxR from Plasmodium falciparum (PfTrxR) is a homodimer with a subunit Mr of 59 000. Each monomer contains one FAD and one redox active disulfide. Despite the high degress of similarity between PfTrxR and the human TrxR, their primary structures present a striking difference in the C-terminus. PfTrxR has two cysteine residues near the C-terminal Gly, while the human TrxR contains a Cys-SeCys dipeptide penultimate to the C-terminal Gly. It has been proposed that the C-terminal cysteines (as a cystine) of PfTrxR are involved in catalysis by an intramolecular dithiol-disulfide interchange with the nascent redox active dithiol. To investigate the proposed function of the C-terminal cysteines of PfTrxR, each has been changed to an alanine [Gilberger, T.-M., Bergmann, B., Walter, R. D., and Müller, S. (1998) FEBS Lett. 425, 407-410]. The single C-terminal cysteine remaining in each mutant was modified with 5,5'-dithiobis(2-nitrobenzoic acid) to form mixed disulfides consisting of the enzyme thiol and thionitrobenzoate (TNB). In reductive titrations of these mixed disulfide enzymes, 1 equiv of TNB anion was released upon reduction of the enzyme itself, while control experiments in which mutants without C-terminal cysteine were used showed little TNB anion release. This suggests that each of the C-terminal cysteines as a TNB mixed disulfide does mimic the proposed electron acceptor in the C-terminus. Analysis of the rapid reaction kinetics showed that the C-terminal mixed disulfide of the modified enzyme is reduced at a rate which is comparable with the turnover number of the wild type enzyme.

Alanine↗
PubMed1999

Crystal structure of reduced thioredoxin reductase from Escherichia coli: structural flexibility in the isoalloxazine ring of the flavin adenine dinucleotide cofactor.

Catalysis by thioredoxin reductase (TrxR) from Escherichia coli requires alternation between two domain arrangements. One of these conformations has been observed by X-ray crystallography (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816). This form of TrxR, denoted FO, permits the reaction of enzyme-bound reduced FAD with a redox-active disulfide on TrxR. As part of an investigation of conformational changes and intermediates in catalysis by TrxR, an X-ray structure of the FO form of TrxR with both the FAD and active site disulfide reduced has been determined. Reduction after crystallization resulted in significant local conformation changes. The isoalloxazine ring of the FAD cofactor, which is essentially planar in the oxidized enzyme, assumes a 34 degree "butterfly" bend about the N(5)-N(10) axis in reduced TrxR. Theoretical calculations reported by others predict ring bending of 15-28 degrees for reduced isoalloxazines protonated at N(1). The large bending in reduced TrxR is attributed in part to steric interactions between the isoalloxazine ring and the sulfur of Cys138, formed by reduction of the active site disulfide, and is accompanied by changes in the positions and interactions of several of the ribityl side-chain atoms of FAD. The bending angle in reduced TrxR is larger than that for any flavoprotein in the Protein Data Bank. Distributions of bending angles in published oxidized and reduced flavoenzyme structures are different from those found in studies of free flavins, indicating that the protein environment has a significant effect on bending.

Amino Acid Sequence↗