Kinetic mechanism of DNA polymerase I (Klenow fragment): identification of a second conformational change and evaluation of the internal equilibrium constant.


:In a previously determined minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment (KF) of Escherichia coli DNA polymerase I, a nonchemical step that interconverted the KF'.DNAn+1.PPi and KF.DNAn+1PPi complexes was not observed in correct incorporation [Kuchta, R. D., Mizrahi, V., Benkovic, P.A., Johnson, K.A., & Benkovic, S.J. (1987) Biochemistry 26, 8410-8417] but was detected in misincorporation [Kuchta, R. D., Benkovic, P.A., & Benkovic, S.J. (1988) Biochemistry 27, 6716-6725]. In a pulse-chase experiment in this study, a burst amplitude of 100% of the enzyme concentration is observed; under pulse-quench conditions, the burst amplitude is 80%, indicative of the accumulation of the KF'.DNA.dNTP species owing to a slow step subsequent to chemical bond formation. This latter step was unequivocally identified by single-turnover pyrophosphorolysis and pyrophosphate-exchange experiments as one interconverting KF'.DNAn+1.PPi and KF.DNAn+1.PPi. The rate constants for this step in both directions were established through the rate constants for processive synthesis and pyrophosphorolysis. Pyrophosphorolysis of a 3'-phosphorothioate DNA duplex confirmed that the large elemental effect observed previously [Mizrahi, V., Henrie, R. N., Marlier, J.F., Johnson, K.A., & Benkovic, S.J. (1985) Biochemistry 24, 4010-4018] in this direction but not in polymerization is due to a marked decrease in the affinity of KF for the phosphorothioate-substituted duplex and not to the chemical step. The combination of the experimentally measured equilibrium constant for the bound KF.DNA species with the collective kinetic measurements further extends previous insights into the dynamics of the polymerization process catalyzed by KF.






Dahlberg ME,Benkovic SJ




Has Abstract


1991-05-21 00:00:00












  • Clustering and dynamics of phototransducer signaling domains revealed by site-directed spin labeling electron paramagnetic resonance on SRII/HtrII in membranes and nanodiscs.

    abstract::In halophilic archaea the photophobic response is mediated by the membrane-embedded 2:2 photoreceptor/-transducer complex SRII/HtrII, the latter being homologous to the bacterial chemoreceptors. Both systems bias the rotation direction of the flagellar motor via a two-component system coupled to an extended cytoplasmi...


    pub_type: 杂志文章


    authors: Orban-Glaß I,Voskoboynikova N,Busch KB,Klose D,Rickert C,Mosslehy W,Roder F,Wilkens V,Piehler J,Engelhard M,Steinhoff HJ,Klare JP

    更新日期:2015-01-20 00:00:00

  • Spin-label detection of hemoglobin-membrane interaction at physiological pH.

    abstract::The interaction between hemoglobin and the cytoplasmic surface of human erythrocyte membranes at physiological pH was studied by monitoring the electron paramagnetic resonance (EPR) signal of spin-labeled membrane ghosts in hemoglobin solutions of various concentrations. The EPR spectra indicate the existence of a sig...


    pub_type: 杂志文章


    authors: Fung LW

    更新日期:1981-12-08 00:00:00

  • Rudimentary phosvitin domain in a minor chicken vitellogenin gene.

    abstract::We have determined the nucleotide sequence and the derived amino acid sequence of the phosphoprotein-encoding region of the chicken vitellogenin III gene. The sequence of this minor vitellogenin could be aligned with exon 22 up to exon 27 of the previously sequenced major vitellogenin II gene (van het Schip et al., 19...


    pub_type: 杂志文章


    authors: Byrne BM,de Jong H,Fouchier RA,Williams DL,Gruber M,Ab G

    更新日期:1989-03-21 00:00:00

  • Altering the RNase H primer grip of human immunodeficiency virus reverse transcriptase modifies cleavage specificity.

    abstract::Recent crystallographic data suggest that conserved residues in the connection subdomain and C-terminal ribonuclease H (RNase H) domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) contact the nascent DNA primer and modulate the trajectory of the template relative to the RNase H catalytic ce...


    pub_type: 杂志文章


    authors: Rausch JW,Lener D,Miller JT,Julias JG,Hughes SH,Le Grice SF

    更新日期:2002-04-16 00:00:00

  • 24-homologated 1,25-dihydroxyvitamin D3 compounds: separation of calcium and cell differentiation activities.

    abstract::A series of 24-homologated 1,25-dihydroxyvitamin D3 compounds have been chemically synthesized and studied with regard to their activity in inducing differentiation of human promyelocyte HL-60 cells to monocytes and in calcium mobilizing activity in vitamin D deficient rats. Homologation of 1,25-dihydroxyvitamin D3 or...


    pub_type: 杂志文章


    authors: Perlman K,Kutner A,Prahl J,Smith C,Inaba M,Schnoes HK,DeLuca HF

    更新日期:1990-01-09 00:00:00

  • Oxy intermediates of homoprotocatechuate 2,3-dioxygenase: facile electron transfer between substrates.

    abstract::Substrates homoprotocatechuate (HPCA) and O(2) bind to the Fe(II) of homoprotocatechuate 2,3-dioxygenase (FeHPCD) in adjacent coordination sites. Transfer of an electron(s) from HPCA to O(2) via the iron is proposed to activate the substrates for reaction with each other to initiate aromatic ring cleavage. Here, rapid...


    pub_type: 杂志文章


    authors: Mbughuni MM,Chakrabarti M,Hayden JA,Meier KK,Dalluge JJ,Hendrich MP,Münck E,Lipscomb JD

    更新日期:2011-11-29 00:00:00

  • Groups with polar characteristics can locate at both shallow and deep locations in membranes: the behavior of dansyl and related probes.

    abstract::To understand the relationship between the chemical structure of polar molecules and their membrane location, the behavior of dansyl (dimethylaminonaphthalenesulfonyl) and related polar fluorescent probes was examined. The depth of these probes in lipid bilayers was determined by parallax analysis of fluorescence quen...


    pub_type: 杂志文章


    authors: Asuncion-Punzalan E,Kachel K,London E

    更新日期:1998-03-31 00:00:00

  • Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding.

    abstract::The preferential interactions of proteins with solvent components were studied in concentrated aqueous solutions of the sulfate, acetate, and chloride salts of Mg2+, Ba2+, Ca2+, Mn2+ and Ni2+ [except for CaSO4, BaSO4, Mn-(OAc)2, and Ni(OAc)2], and results were compared with those of the Na+ salts. It was found that, f...


    pub_type: 杂志文章


    authors: Arakawa T,Timasheff SN

    更新日期:1984-12-04 00:00:00

  • Optical spectroscopy of nicotinoprotein alcohol dehydrogenase from Amycolatopsis methanolica: a comparison with horse liver alcohol dehydrogenase and UDP-galactose epimerase.

    abstract::The NADH absorbance spectrum of nicotinoprotein (NADH-containing) alcohol dehydrogenase from Amycolatopsis methanolica has a maximum at 326 nm. Reduced enzyme-bound pyridine dinucleotide could be reversibly oxidized by acetaldehyde. The fluorescence excitation spectrum for NADH bound to the enzyme has a maximum at 325...


    pub_type: 杂志文章


    authors: Piersma SR,Visser AJ,de Vries S,Duine JA

    更新日期:1998-03-03 00:00:00

  • Deletion of the Mitochondrial Protein VWA8 Induces Oxidative Stress and an HNF4α Compensatory Response in Hepatocytes.

    abstract::von Willebrand A domain-containing protein 8 (VWA8) is a poorly characterized, mitochondrial matrix-targeted protein with an AAA ATPase domain and ATPase activity that increases in livers of mice fed a high-fat diet. This study was undertaken to use CRISPR/Cas9 to delete VWA8 in cultured mouse hepatocytes and gain ins...


    pub_type: 杂志文章


    authors: Luo M,Willis WT,Coletta DK,Langlais PR,Mengos A,Ma W,Finlayson J,Wagner GR,Shi CX,Mandarino LJ

    更新日期:2019-12-10 00:00:00

  • Structural characterization of folded and unfolded states of an SH3 domain in equilibrium in aqueous buffer.

    abstract::The isolated N-terminal Src homology 3 (SH3) domain of Drosophila drk exists in equilibrium between folded and unfolded states in aqueous buffer near neutral pH. Nuclear magnetic resonance spectra recorded on both states simultaneously exhibit an approximate 1:1 ratio of protein conformations. The folded form is simil...


    pub_type: 杂志文章


    authors: Zhang O,Forman-Kay JD

    更新日期:1995-05-23 00:00:00

  • Characterization of a small metal binding protein from Nitrosomonas europaea.

    abstract::A small metal-binding protein (SmbP) with no known similarity to other proteins in current databases was isolated and characterized from the periplasm of Nitrosomonas europaea. The primary structure of this small (9.9 kDa) monomeric protein is characterized by a series of 10 repeats of a seven amino acid motif and an ...


    pub_type: 杂志文章


    authors: Barney BM,LoBrutto R,Francisco WA

    更新日期:2004-09-07 00:00:00

  • Functional segregation of a predicted "hinge" site within the beta-strand linkers of Escherichia coli leucyl-tRNA synthetase.

    abstract::Some aminoacyl-tRNA synthetases (AARSs) employ an editing mechanism to ensure the fidelity of protein synthesis. Leucyl-tRNA synthetase (LeuRS), isoleucyl-tRNA synthetase (IleRS), and valyl-tRNA synthetase (ValRS) share a common insertion, called the CP1 domain, which is responsible for clearing misformed products. Th...


    pub_type: 杂志文章


    authors: Mascarenhas AP,Martinis SA

    更新日期:2008-04-22 00:00:00

  • Partitioning of synaptotagmin I C2 domains between liquid-ordered and liquid-disordered inner leaflet lipid phases.

    abstract::Synaptotagmin I is the calcium sensor in synchronous neurotransmitter release caused by fusion of synaptic vesicles with the presynaptic membrane in neurons. Synaptotagmin I interacts with acidic phospholipids, but also with soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs), at various stages in ...


    pub_type: 杂志文章


    authors: Wan C,Kiessling V,Cafiso DS,Tamm LK

    更新日期:2011-04-05 00:00:00

  • Mechanism of interactions of alpha-naphthoflavone with cytochrome P450 3A4 explored with an engineered enzyme bearing a fluorescent probe.

    abstract::Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into alpha-helix A. The site of modification was identified as Cys-64 with the help of CYP3A4(C58) and CYP3A4(C64), each bearing ...


    pub_type: 杂志文章


    authors: Tsalkova TN,Davydova NY,Halpert JR,Davydov DR

    更新日期:2007-01-09 00:00:00

  • Complementary DNA cloning and kinetic characterization of a novel intracellular serine proteinase inhibitor: mechanism of action with trypsin and factor Xa as model proteinases.

    abstract::The full-length cDNA encoding a novel human intracellular serine proteinase inhibitor has been sequenced and found to encode a 376 amino acid protein (M(r) approximately 42.5K) that we designate as cytoplasmic antiproteinase. Analysis of the primary structure revealed that the cytoplasmic antiproteinase has the majori...


    pub_type: 杂志文章


    authors: Morgenstern KA,Sprecher C,Holth L,Foster D,Grant FJ,Ching A,Kisiel W

    更新日期:1994-03-22 00:00:00

  • Evaluation of copper2+ affinities for the prion protein.

    abstract::The prion protein (PrP) is a cell-surface Cu2+ binding glycoprotein which can bind six copper ions. The role of Cu2+ in PrP function, misfolding, and prion disease has generated much interest; however, the field has been hampered by a lack of consensus with regard to the affinity of Cu2+ for PrPC. Here we build on our...


    pub_type: 杂志文章


    authors: Nadal RC,Davies P,Brown DR,Viles JH

    更新日期:2009-09-29 00:00:00

  • Cardiac troponin T forms a tetramer in vitro.

    abstract::Cardiac troponin T (cTnT) is a myofibrillar protein essential for calcium-dependent contraction. This property has led to functional studies of developmentally expressed cTnT isoforms and mutants identified in patients with hypertrophic cardiomyopathy. The release of cTnT into the serum following myocardial infarction...


    pub_type: 杂志文章


    authors: Lounes KC,Demeler B,Anderson DE,Gomes AW,Potter JD,Nassar R,Anderson PA

    更新日期:2008-02-19 00:00:00

  • Spectroscopic Determination of Distinct Heme Ligands in Outer-Membrane Receptors PhuR and HasR of Pseudomonas aeruginosa.

    abstract::Pseudomonas aeruginosa PAO1 encodes two outer membrane receptors, PhuR (Pseudomonas heme uptake) and HasR (heme assimilation system). The HasR receptor acquires heme through interaction with a secreted hemophore, HasAp. The non-hemophore-dependent PhuR is encoded along with proteins required for heme translocation int...


    pub_type: 杂志文章


    authors: Smith AD,Modi AR,Sun S,Dawson JH,Wilks A

    更新日期:2015-04-28 00:00:00

  • Extracellular labeling of growing secreted polypeptide chains in Bacillus subtilis with diazoiodosulfanilic acid.

    abstract::Studies of the mechanism of protein secretion in a Gram-positive bacterium, Bacillus subtilis, yielded results very similar to those previously obtained with a Gram-negative organism: nascent chains protruding from protoplasts could be labeled extracellularly; the labeled chains could be recovered on polysomes isolate...


    pub_type: 杂志文章


    authors: Smith WP,Tai PC,Davis BD

    更新日期:1979-01-09 00:00:00

  • Homogeneity and variability in the structure of azurin molecules studied by fluorescence decay and circular polarization.

    abstract::The fluorescence decay of apoazurin derived from Pseudomonas aeruginosa is monoexponential. By this criterion the population of molecules of apoazurin is homogeneous. The emission anisotropy factor and the absorption anisotropy factor at the red edge of the absorption band assume similar values, showing that the trypt...


    pub_type: 杂志文章


    authors: Grinvald A,Schlessinger J,Pecht I,Steinberg IZ

    更新日期:1975-05-06 00:00:00

  • Influence of iron-removal procedures on sequential electron transfer in photosynthetic bacterial reaction centers studied by transient EPR spectroscopy.

    abstract::Electron spin polarized electron paramagentic resonance (ESP EPR) spectra were obtained with deuterated iron-removed photosynthetic bacterial reaction centers (RCs) to specifically investigate the effect of the rate of primary charge separation, metal-site occupancy, and H-subunit content on the observed P865+QA- char...


    pub_type: 杂志文章


    authors: Utschig LM,Greenfield SR,Tang J,Laible PD,Thurnauer MC

    更新日期:1997-07-15 00:00:00

  • Pulsed electron paramagnetic resonance studies of the lysine 2,3-aminomutase substrate radical: evidence for participation of pyridoxal 5'-phosphate in a radical rearrangement.

    abstract::The role of pyridoxal 5'-phosphate (PLP) in the radical-mediated amino group migration catalyzed by lysine 2,3-aminomutase from Clostridia SB4 has been investigated by electron spin echo envelope modulation (ESEEM) spectroscopy. This pulsed electron paramagnetic resonance (EPR) method was used to estimate the distance...


    pub_type: 杂志文章


    authors: Ballinger MD,Frey PA,Reed GH,LoBrutto R

    更新日期:1995-08-08 00:00:00

  • Crystallographic studies of isosteric NAD analogues bound to alcohol dehydrogenase: specificity and substrate binding in two ternary complexes.

    abstract::CNAD (5-beta-D-ribofuranosylnicotinamide adenine dinucleotide) is an isosteric C-glycosidic analogue of NAD(H) containing a neutral pyridine ring. CPAD (5-beta-D-ribofuranosylpicolinamide adenine dinucleotide) is a closely related pyridine-containing analogue with the pyridine nitrogen on the opposite side of the ring...


    pub_type: 杂志文章


    authors: Li H,Hallows WH,Punzi JS,Pankiewicz KW,Watanabe KA,Goldstein BM

    更新日期:1994-10-04 00:00:00

  • Self-assembly of designed antimicrobial peptides in solution and micelles.

    abstract::Hydrophobic interactions are responsible for stabilizing leucine zippers in peptides containing heptad repeats. The effects of substituting leucine by phenylalanine and alanine by glycine on the self-assembly of coiled-coils were examined in minimalist antimicrobial peptides designed to form amphipathic alpha-helices....


    pub_type: 杂志文章


    authors: Javadpour MM,Barkley MD

    更新日期:1997-08-05 00:00:00

  • Effects of the Iowa and Milano mutations on apolipoprotein A-I structure and dynamics determined by hydrogen exchange and mass spectrometry.

    abstract::The Iowa point mutation in apolipoprotein A-I (G26R) leads to a systemic amyloidosis condition, and the Milano mutation (R173C) is associated with hypoalphalipoproteinemia, a reduced plasma level of high-density lipoprotein. To probe the structural effects that lead to these outcomes, we used amide hydrogen-deuterium ...


    pub_type: 杂志文章


    authors: Chetty PS,Ohshiro M,Saito H,Dhanasekaran P,Lund-Katz S,Mayne L,Englander W,Phillips MC

    更新日期:2012-11-06 00:00:00

  • Structure and enzymatic functions of thioredoxin refolded by complementation of two tryptic peptide fragments.

    abstract::The physicochemical and catalytic properties of thioredoxin-T' are described. This complemented protein structure consists of a 1:1 complex between the inactive fragments thioredoxin-T-(1--73) and thioredoxin T-(74--108). These are generated by selective trypsin cleavage at Arg-73 in lysine-modified and denatured Esch...


    pub_type: 杂志文章


    authors: Slabý I,Holmgren A

    更新日期:1979-12-11 00:00:00

  • Allosteric regulation in phosphofructokinase from the extreme thermophile Thermus thermophilus.

    abstract::An investigation into the kinetics and regulatory properties of the type-1 phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus (TtPFK) reveals an enzyme that is inhibited by PEP and activated by ADP by modifying the affinity exhibited for the substrate fructose 6-phosphate (Fru-6-P) in a manner...


    pub_type: 杂志文章


    authors: McGresham MS,Lovingshimer M,Reinhart GD

    更新日期:2014-01-14 00:00:00

  • Pre-steady-state kinetics reveal a slow isomerization of the enzyme-NAD complex in the NAD-malic enzyme reaction.

    abstract::Stopped-flow experiments obtained in the pre-steady-state time scale of the NAD-malic enzyme reaction exhibit a lag prior to the attainment of steady state. Previous results from isotope effect studies in which the deuterium isotope effect on Vmax decreases to a value of 1 at low pH have been interpreted as suggesting...


    pub_type: 杂志文章


    authors: Rajapaksa R,Abu-Soud H,Raushel FM,Harris BG,Cook PF

    更新日期:1993-03-02 00:00:00

  • Excessive Reactive Oxygen Species and Exotic DNA Lesions as an Exploitable Liability.

    abstract::Although the terms "excessive reactive oxygen species (ROS)" and "oxidative stress" are widely used, the implications of oxidative stress are often misunderstood. ROS are not a single species but a variety of compounds, each with unique biochemical properties and abilities to react with biomolecules. ROS cause activat...


    pub_type: 杂志文章,评审


    authors: AbdulSalam SF,Thowfeik FS,Merino EJ

    更新日期:2016-09-27 00:00:00