Effects of temperature and viscosity on R67 dihydrofolate reductase catalysis.


:R67 dihydrofolate reductase (DHFR) is a novel homotetrameric protein that possesses 222 symmetry and a single, voluminous active site pore. This symmetry poses numerous limitations on catalysis; for example, two dihydrofolate (DHF) molecules or two NADPH molecules, or one substrate plus one cofactor can bind. Only the latter combination leads to catalysis. To garner additional information on how this enzyme facilitates transition-state formation, the temperature dependence of binding and catalysis was monitored. The binding of NADPH and DHF is enthalpy-driven. Previous primary isotope effect studies indicate hydride transfer is at least partially rate-determining. Accordingly, the activation energy associated with transition-state formation was measured and is found to be 6.9 kcal/mol (DeltaH(++)(25) = 6.3 kcal/mol). A large entropic component is also found associated with catalysis, TDeltaS(++)(25) = -11.3 kcal/mol. The poor substrate, dihydropteroate, binds more weakly than dihydrofolate (DeltaDeltaG = 1.4 kcal/mol) and displays a large loss in the binding enthalpy value (DeltaDeltaH = 3.8 kcal/mol). The k(cat) value for dihydropteroate reduction is decreased 1600-fold compared to DHF usage. This effect appears to derive mostly from the DeltaDeltaH difference in binding, demonstrating that the glutamate tail is important for catalysis. This result is surprising, as the para-aminobenzoyl-glutamate tail of DHF has been previously shown to be disordered by both NMR and crystallography studies. Viscosity studies were also performed and confirmed that the hydride transfer rate is not sensitive to sucrose addition. Surprisingly, binding of DHF, by both K(m) and K(d) determination, was found to be sensitive to added viscogens, suggesting a role for water in DHF binding.






Chopra S,Lynch R,Kim SH,Jackson M,Howell EE




Has Abstract


2006-05-30 00:00:00












  • Affinity cleavage at the metal-binding site of phosphoenolpyruvate carboxykinase.

    abstract::Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at pH 7.4. Omitting ascorbate or replacing the Fe2+ with Mn2+ or Mg2+ gives no inactivation. Mn2+, Mg2+, or Co2+ at 100-fold molar excess over Fe2+ offered comple...


    pub_type: 杂志文章


    authors: Hlavaty JJ,Nowak T

    更新日期:1997-12-09 00:00:00

  • Expression and structural and functional properties of human ferritin L-chain from Escherichia coli.

    abstract::The human ferritin L-chain cDNA was cloned into a vector for overproduction in Escherichia coli, under the regulation of a lambda promoter. The plasmid obtained contains the full L-chain coding region modified at the first two codons. It is able to direct the synthesis of the L-chain which can constitute up to 15% of ...


    pub_type: 杂志文章


    authors: Levi S,Salfeld J,Franceschinelli F,Cozzi A,Dorner MH,Arosio P

    更新日期:1989-06-13 00:00:00

  • Site specific combinations of stabilizing and destabilizing amino acid replacements in yeast cytochrome c: in vivo and in vitro effects.

    abstract::Oligonucleotide-directed mutagenesis in vivo was used to create destabilizing site specific changes at position 6 in iso-1-cytochrome c in Saccharomyces cerevisiae. These changes were made in combination with the stabilizing N52I substitution. The resulting proteins showed that a variety of forces and interactions are...


    pub_type: 杂志文章


    authors: Linske-O'Connell LI,Sherman F,McLendon G

    更新日期:1995-05-30 00:00:00

  • Activity-dependent phosphorylation of dynamin 1 at serine 857.

    abstract::Dynamin 1 is thought to mediate synaptic transmission through interactions with multiple endocytic accessory proteins in a phosphorylation-dependent manner. Previously, we have shown that DYRK1A, a chromosome 21-encoded kinase implicated in the mental retardation of Down syndrome, phosphorylates primarily serine 857 (...


    pub_type: 杂志文章


    authors: Xie W,Adayev T,Zhu H,Wegiel J,Wieraszko A,Hwang YW

    更新日期:2012-08-28 00:00:00

  • Kinetic studies of the branched chain amino acid preferring peptidase activity of the 20S proteasome: development of a continuous assay and inhibition by tripeptide aldehydes and clasto-lactacystin beta-lactone.

    abstract::We have developed an assay to continuously monitor the branched amino acid preferring peptidase (BrAAP) activity of the proteasome. This assay is based on the hydrolysis of the fluorogenic peptide, Abz-Gly-Pro-Ala-Leu-Ala-Nba (Abz is 2-aminobenzoyl and Nba is 4-nitrobenzylamide) which is cleaved exclusively at the Leu...


    pub_type: 杂志文章


    authors: McCormack TA,Cruikshank AA,Grenier L,Melandri FD,Nunes SL,Plamondon L,Stein RL,Dick LR

    更新日期:1998-05-26 00:00:00

  • Secondary binding sites for triplex-forming oligonucleotides containing bulges, loops, and mismatches in the third strand.

    abstract::We have used DNase I footprinting to examine the binding of five different 17-mer oligonucleotides to a 53-base oligopurine tract containing four pyrimidine interruptions. Although all the expected triplexes formed with high affinity (K(d) approximately 10-50 nM), one oligonucleotide produced a footprint at a second s...


    pub_type: 杂志文章


    authors: Fox KR,Flashman E,Gowers D

    更新日期:2000-06-06 00:00:00

  • Interactions of oleic acid with liver fatty acid binding protein: a carbon-13 NMR study.

    abstract::13C NMR spectroscopy was used to probe the structural interactions between carboxyl-13C-enriched oleic acid (18:1) and rat liver fatty acid binding protein (FABP) and the partitioning of 18:1 between FABP and unilamellar phosphatidylcholine (PC) vesicles. Spectra of systems containing 2-8 mol of 18:1/mol of FABP (but ...


    pub_type: 杂志文章


    authors: Cistola DP,Walsh MT,Corey RP,Hamilton JA,Brecher P

    更新日期:1988-01-26 00:00:00

  • A new N-terminal recognition domain in caveolin-1 interacts with sterol carrier protein-2 (SCP-2).

    abstract::Although plasma membrane domains, such as caveolae, provide an organizing principle for signaling pathways and cholesterol homeostasis in the cell, relatively little is known regarding specific mechanisms, whereby intracellular lipid-binding proteins are targeted to caveolae. Therefore, the interaction between caveoli...


    pub_type: 杂志文章


    authors: Parr RD,Martin GG,Hostetler HA,Schroeder ME,Mir KD,Kier AB,Ball JM,Schroeder F

    更新日期:2007-07-17 00:00:00

  • Purification of recombinant pp60v-src protein tyrosine kinase and phosphorylation of peptides with different secondary structure preference.

    abstract::The expression of the transforming gene product of Rous sarcoma virus (pp60v-src) in Saccharomyces cerevisiae has recently been reported (Kornbluth et al., 1987; Brugge et al., 1987). To carry out biochemical and structural studies of this enzyme, a facile purification was developed. The purification was accomplished ...


    pub_type: 杂志文章


    authors: Radziejewski C,Miller WT,Mobashery S,Goldberg AR,Kaiser ET

    更新日期:1989-11-14 00:00:00

  • Acid- and calcium-induced structural changes in phosphatidylethanolamine membranes stabilized by cholesteryl hemisuccinate.

    abstract::The membrane stabilization effect of cholesteryl hemisuccinate (CHEMS) and the sensitivity of the CHEMS-phosphatidylethanolamine membranes to protons and calcium ions were studied by differential scanning calorimetry, freeze-fracture electron microscopy, and 31P NMR. (1) At neutral pH, the addition of 8 mol % CHEMS to...


    pub_type: 杂志文章


    authors: Lai MZ,Vail WJ,Szoka FC

    更新日期:1985-03-26 00:00:00

  • Continuous flow reactor for the production of stable amyloid protein oligomers.

    abstract::The predominant working hypothesis of Alzheimer's disease is that the proximate pathologic agents are oligomers of the amyloid β-protein (Aβ). "Oligomer" is an ill-defined term. Many different types of oligomers have been reported, and they often exist in rapid equilibrium with monomers and higher-order assemblies. Th...


    pub_type: 杂志文章


    authors: Hayden EY,Teplow DB

    更新日期:2012-08-14 00:00:00

  • A reexamination of the reported B----Z DNA transition in nucleosomes reconstituted with poly(dG-m5dC).poly(dG-m5dC).

    abstract::Polynucleosomes with poly(dG-m5dC).poly(dG-m5dC) have been reconstituted, and well-defined nucleosome core particles from these have been prepared. Upon addition of MgCl2 to the levels used to induce the B to Z transition in this highly methylated DNA, significant changes in the circular dichroism spectrum are observe...


    pub_type: 杂志文章


    authors: Ausio J,Zhou G,van Holde K

    更新日期:1987-09-08 00:00:00

  • Crystal structure of human fibrinogen.

    abstract::A crystal structure of human fibrinogen has been determined at approximately 3.3 A resolution. The protein was purified from human blood plasma, first by a cold ethanol precipitation procedure and then by stepwise chromatography on DEAE-cellulose. A product was obtained that was homogeneous on SDS-polyacrylamide gels....


    pub_type: 杂志文章


    authors: Kollman JM,Pandi L,Sawaya MR,Riley M,Doolittle RF

    更新日期:2009-05-12 00:00:00

  • Loss of DNA minor groove interactions by exonuclease-deficient Klenow polymerase inhibits O6-methylguanine and abasic site translesion synthesis.

    abstract::The importance of DNA polymerase-DNA minor groove interactions on translesion synthesis (TLS) was examined in vitro using variants of exonuclease-deficient Klenow polymerase and site-specifically modified DNA oligonucleotides. Polymerase variant R668A lacks primer strand interactions, while variant Q849A lacks templat...


    pub_type: 杂志文章


    authors: Gestl EE,Eckert KA

    更新日期:2005-05-10 00:00:00

  • Identification of two serine residues essential for agonist-induced 5-HT2A receptor desensitization.

    abstract::5-HT(2A) serotonin receptors represent the principal molecular targets for LSD-like hallucinogens and atypical antipsychotic drugs. It has been proposed that a dysregulation of 5-HT(2A) receptor-mediated signaling may contribute to the pathogenesis of schizophrenia and related diseases. A major mechanism for the atten...


    pub_type: 杂志文章


    authors: Gray JA,Compton-Toth BA,Roth BL

    更新日期:2003-09-16 00:00:00

  • Molecular dynamics simulations of binding modes between methylene blue and DNA with alternating GC and AT sequences.

    abstract::The understanding of interactions between small molecules and DNA is crucial to design new anticancer drugs targeted to DNA. Methylene blue (MB) is a phenothiazinium dye that has shown promising results in photodynamic therapy treatment. The noncovalent binding of methylene blue to DNA was experimentally and theoretic...


    pub_type: 杂志文章


    authors: Nogueira JJ,González L

    更新日期:2014-04-15 00:00:00

  • Properties of overlapping EREs: synergistic activation of transcription and cooperative binding of ER.

    abstract::We have designed a novel estrogen-responsive unit, overERE, which consists of two overlapping ERE separated by 5 bp (center-to-center). In gel retardation assays, this sequence forms a low-mobility complex that migrates like an estrogen receptor tetramer. The receptor-overERE complex was specific and was supershifted ...


    pub_type: 杂志文章


    authors: Massaad C,Coumoul X,Sabbah M,Garlatti M,Redeuilh G,Barouki R

    更新日期:1998-04-28 00:00:00

  • Subtle Dynamic Changes Accompany Hck Activation by HIV-1 Nef and are Reversed by an Antiretroviral Kinase Inhibitor.

    abstract::The HIV-1 virulence factor Nef interacts with the macrophage Src-family kinase Hck, resulting in constitutive kinase activation that contributes to viral replication and immune escape. Previous chemical library screens identified the diphenylfuranopyrimdine kinase inhibitor DFP-4AB, which selectively inhibits Nef-depe...


    pub_type: 杂志文章


    authors: Wales TE,Hochrein JM,Morgan CR,Emert-Sedlak LA,Smithgall TE,Engen JR

    更新日期:2015-10-20 00:00:00

  • Amino acid structures of multiple forms of amyloid-related serum protein SAA from a single individual.

    abstract::Multiple forms of the acute-phase serum protein SAA were isolated from the lipoprotein fraction of plasma from a single individual. These protein forms were purified by size-exclusion, ion-exchange, and reverse-phase high-pressure liquid chromatography, and then the tryptic peptides were subjected to amino acid sequen...


    pub_type: 杂志文章


    authors: Dwulet FE,Wallace DK,Benson MD

    更新日期:1988-03-08 00:00:00

  • Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helices IV and V that contain the major determinants for substrate binding.

    abstract::Helices IV and V in the lactose permease of Escherichia coli contain the major determinants for substrate binding [Glu126 (helix IV), Arg144 (helix V), and Cys148 (helix V)]. Structural and dynamic features of this region were studied by using site-directed sulfhydryl modification of 48 single-Cys replacement mutants ...


    pub_type: 杂志文章


    authors: Kwaw I,Zen KC,Hu Y,Kaback HR

    更新日期:2001-09-04 00:00:00

  • Human frataxin activates Fe-S cluster biosynthesis by facilitating sulfur transfer chemistry.

    abstract::Iron-sulfur clusters are ubiquitous protein cofactors with critical cellular functions. The mitochondrial Fe-S assembly complex, which consists of the cysteine desulfurase NFS1 and its accessory protein (ISD11), the Fe-S assembly protein (ISCU2), and frataxin (FXN), converts substrates l-cysteine, ferrous iron, and el...


    pub_type: 杂志文章


    authors: Bridwell-Rabb J,Fox NG,Tsai CL,Winn AM,Barondeau DP

    更新日期:2014-08-05 00:00:00

  • Mechanistic insight into the photosensory versatility of DXCF cyanobacteriochromes.

    abstract::Cyanobacteriochromes (CBCRs) are photosensory proteins related to the red/far-red phytochromes. Like phytochromes, CBCRs use linear tetrapyrrole (bilin) chromophores covalently attached via a thioether linkage to a conserved Cys residue also found in plant and cyanobacterial phytochromes. Unlike almost all phytochrome...


    pub_type: 杂志文章


    authors: Rockwell NC,Martin SS,Lagarias JC

    更新日期:2012-05-01 00:00:00

  • Predominant interactions between mu-conotoxin Arg-13 and the skeletal muscle Na+ channel localized by mutant cycle analysis.

    abstract::High-affinity mu-conotoxin block of skeletal muscle Na+ channels depends on an arginine at position 13 (Arg-13). To understand both the mechanism of toxin interaction and the general structure of its binding site in the channel mouth, we examined by thermodynamic mutant cycle analysis the interaction between the criti...


    pub_type: 杂志文章


    authors: Chang NS,French RJ,Lipkind GM,Fozzard HA,Dudley S Jr

    更新日期:1998-03-31 00:00:00

  • Bovine dentin phosphophoryn: composition and molecular weight.

    abstract::The molecular weight of phosphophoryn, an acidic phosphoprotein unique to dentin matrix, has been difficult to determine because of a combination of neutral protease activities in this tissue and the intrinsic high charge density of the molecule. In this study, bovine dentin phosphophoryn (BDPP) was isolated by a proc...


    pub_type: 杂志文章


    authors: Stetler-Stevenson WG,Veis A

    更新日期:1983-08-30 00:00:00

  • Structure, topology, and tilt of cell-signaling peptides containing nuclear localization sequences in membrane bilayers determined by solid-state NMR and molecular dynamics simulation studies.

    abstract::Cell-signaling peptides have been extensively used to transport functional molecules across the plasma membrane into living cells. These peptides consist of a hydrophobic sequence and a cationic nuclear localization sequence (NLS). It has been assumed that the hydrophobic region penetrates the hydrophobic lipid bilaye...


    pub_type: 杂志文章


    authors: Ramamoorthy A,Kandasamy SK,Lee DK,Kidambi S,Larson RG

    更新日期:2007-01-30 00:00:00

  • Competition between Different S-Components for the Shared Energy Coupling Factor Module in Energy Coupling Factor Transporters.

    abstract::Energy coupling factor (ECF) transporters take up micronutrients in Bacteria and Archaea. They consist of a membrane-embedded S-component that provides substrate specificity and a three-subunit ECF module that couples ATP hydrolysis to transport. The S-components ThiT (for thiamin) and NiaX (for niacin) from Lactococc...


    pub_type: 杂志文章


    authors: Majsnerowska M,Ter Beek J,Stanek WK,Duurkens RH,Slotboom DJ

    更新日期:2015-08-11 00:00:00

  • A 106-kDa aminopeptidase is a putative receptor for Bacillus thuringiensis Cry11Ba toxin in the mosquito Anopheles gambiae.

    abstract::Bacillus thuringiensis (Bt) insecticidal toxins bind to receptors on midgut epithelial cells of susceptible insects, and binding triggers biochemical events that lead to insect mortality. Recently, a 100-kDa aminopeptidase N (APN) was isolated from brush border membrane vesicles (BBMV) of Anopheles quadrimaculatus and...


    pub_type: 杂志文章


    authors: Zhang R,Hua G,Andacht TM,Adang MJ

    更新日期:2008-10-28 00:00:00

  • Identification of competitive antagonists of the rod photoreceptor cGMP-gated cation channel: beta-phenyl-1,N2-etheno-substituted cGMP analogues as probes of the cGMP-binding site.

    abstract::cGMP is the natural activator of the cyclic nucleotide-gated channel originally isolated from rod photoreceptors but now known to be expressed in a wide variety of neural and non-neural cells. To identify antagonists of cGMP action and to better understand the interaction between cGMP and the channel protein, experime...


    pub_type: 杂志文章


    authors: Wei JY,Cohen ED,Yan YY,Genieser HG,Barnstable CJ

    更新日期:1996-12-24 00:00:00

  • Binding of proteins to specific target sites in membranes measured by total internal reflection fluorescence microscopy.

    abstract::A new quantitative technique for measuring the binding of proteins to membranes is described. The method is based on a combination of total internal reflection fluorescence microscopy and the preparation of supported planar bilayers. Specific and reversible binding of a fluorescence-labeled monoclonal antibody to lipi...


    pub_type: 杂志文章


    authors: Kalb E,Engel J,Tamm LK

    更新日期:1990-02-13 00:00:00

  • Photo-cross-linking of CRP to nonspecific DNA in the absence of cAMP. DNA interacts with both the N- and C-terminal parts of the protein.

    abstract::Adenosine cyclic 3',5'-phosphate receptor protein (CRP or CAP) is a regulatory protein involved in the transcription of several operons in Escherichia coli. cAMP-independent, nonspecific complexes of CRP and DNA were investigated by photochemical cross-linking of the protein to nonspecific DNA, whose thymines are subs...


    pub_type: 杂志文章


    authors: Katouzian-Safadi M,Blazy B,Cremet JY,Le Caer JP,Rossier J,Charlier M

    更新日期:1993-02-23 00:00:00