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Douglas B. Craig - One of the best experts on this subject based on the ideXlab platform.

  • Determination of the inhibitor dissociation constant of an individual unmodified Enzyme Molecule in free solution
    Electrophoresis, 2016
    Co-Authors: Jeremie J. Crawford, Joshua W. Hollett, Douglas B. Craig
    Abstract:

    Single Enzyme Molecule assays on E. coli β-galactosidase were performed using a capillary electrophoresis-based method. Three types of assays were performed. The catalytic rate of 20 individual Molecules was assayed in duplicate in the presence of 50 μM substrate. The ratio of rates for the second incubation relative to the first was 0.96 ± 0.03, showing the reproducibility of the method. In the second assay, the rates were determined in the absence and presence of 210 μM L-ribose, a competitive inhibitor. The ratio of the rate in the presence of inhibitor to that in its absence for 19 individual Molecules was 0.44 ± 0.23. This large relative standard deviation suggests that each individual Enzyme Molecule was affected to a different extent by the presence of the inhibitor, which is consistent with KI being heterogeneous. To estimate KI for individual Molecules, a third assay was performed. Each Molecule was incubated in the presence of 30 and 50 μM substrate and then in the presence of 50 μM substrate plus 210 μM inhibitor. Comparison of the rates in the two substrate concentrations allowed for the determination of the individual Km of each Molecule. From this value and the difference in rates in the presence and absence of inhibitor, the individual Molecule KI values were determined. This value was found to differ between individual Molecules and was found to increase with an increase in Km . Modeling showed that a heterogeneity in KI results in an alteration in the Michaelis-Menten curve for a population of Enzymes in the presence of a competitive inhibitor.

  • Arrhenius plot for a reaction catalyzed by a single Molecule of β-galactosidase.
    Analytical chemistry, 2012
    Co-Authors: Douglas B. Craig, Linden N Chase
    Abstract:

    The activity of a single Enzyme Molecule of Escherichia coli β-galactosidase was measured using a capillary electrophoresis continuous flow assay. As the Enzyme Molecule traversed the capillary the incubation temperature was increased from 27 to 37 °C, providing a continuous record of the change in rate with temperature. This data was used to develop a single Enzyme Molecule Arrhenius plot, from which the activation energy of the reaction was determined to be 31 kJ mol(-1).

  • Single Molecule Assay of Escherichia coli β-Galactosidase Using Two Competing Substrates Simultaneously, DDAO-β-D-Galactoside and Resorufin-β-D-Galactoside
    Analytical Letters, 2011
    Co-Authors: Douglas B. Craig, Michael J. Eggertson, Miki Chikamatsu, Corie A. Horwood
    Abstract:

    Single Enzyme Molecule assays were performed on E. coli β-galactosidase using a capillary electrophoresis-based protocol. Assays were performed using double incubations and two substrates, resorufin-β-D-galactoside and DDAO-β-D-galactoside, simultaneously. The variation between individual Enzyme Molecules in the ratio of product peak areas for the two different substrates used was indistinguishable from the variation in peak areas of the replicate incubations for a given Enzyme Molecule. This suggests that the Enzyme is not heterogeneous with respect to its relative activity with the two different substrates used.

  • Differences in the Average Single Molecule Activities of E. coli β-Galactosidase: Effect of Source, Enzyme Molecule Age and Temperature of Induction
    Journal of protein chemistry, 2003
    Co-Authors: Douglas B. Craig, Juli T. Nachtigall, Heather L. Ash, Glen K. Shoemaker, Ashley C. Dyck, Teresa M. J. Wawrykow, Holly L. Gudbjartson
    Abstract:

    Using a capillary electrophoresis–based method, single Enzyme Molecule assays were performed on E. coli β-galactosidase from three different sets of samples. The first set consisted of lysates of induced cells from five different strains of the bacteria, as well as two different commercial preparations of the Enzyme. These samples were found to have substantially different distributions of single Molecule activities. For the second set of samples, β-galactosidase expression was induced for 1.5 hr, followed by further incubation where expression was repressed. Assays were performed on the lysates of the preinduction and on the lysates from aliquots taken set times postinduction. The recently induced Enzyme had a 25% higher average single Molecule activity than the basally expressed Enzyme. This average activity returned to the basal value 3.5 hr postinduction and remained unchanged thereafter. Finally, β-galactosidase was induced at 26 and 42°C. The Enzyme was assayed before and after partial thermal denaturation. The samples were found to be indistinguishable with respect to their average single Molecule activities.

  • Peer Reviewed: Life and Death of a Single Enzyme Molecule
    Analytical Chemistry, 1998
    Co-Authors: Douglas B. Craig, Edgar A. Arriaga, Jerome C. Y. Wong, And Norman J. Dovichi
    Abstract:

    Details of Enzyme behavior, previously hidden in the ensemble average of classic methods, are revealed by studying individual Molecules.

And Norman J. Dovichi - One of the best experts on this subject based on the ideXlab platform.

Allen J. Bard - One of the best experts on this subject based on the ideXlab platform.

  • Toward single Enzyme Molecule electrochemistry.
    ACS nano, 2008
    Co-Authors: Allen J. Bard
    Abstract:

    Single-Molecule studies, including those of single Enzyme Molecules, have led to important new insights about the effects of environment and configuration on the behavior of these Molecules. Such information is not available from ensemble studies. Most of these have been based on spectroscopic approaches, but there have been relatively few attempts at single-Molecule electrochemistry. In a paper in this issue, progress toward studying a single Enzyme Molecule by protein film voltammetry is described. This Perspective reviews briefly past work on nanoelectrodes and electrochemical and spectroelectroelectrochemical single-Molecule studies, as well as examples of the type of information obtained in past studies of Enzymes.

David R. Walt - One of the best experts on this subject based on the ideXlab platform.

  • Distinct and long-lived activity states of single Enzyme Molecules.
    Journal of the American Chemical Society, 2008
    Co-Authors: David M. Rissin, Hans H. Gorris, David R. Walt
    Abstract:

    Individual Enzyme Molecules have been observed to possess discrete and different turnover rates due to the presence of long-lived activity states. These stable activity states are thought to result from different molecular conformations or post-translational modifications. The distributions in kinetic activity observed in previous studies were obtained from small numbers of single Enzyme Molecules. Due to this limitation, it has not been possible to fully characterize the different kinetic and equilibrium binding parameters of single Enzyme Molecules. In this paper, we analyze hundreds of single β-galactosidase Molecules simultaneously; using a high-density array of 50 000 fL-reaction chambers, we confirm the presence of long-lived kinetic states within a population of Enzyme Molecules. Our analysis has isolated the source of kinetic variability to kcat. The results explain the kinetic variability within Enzyme Molecule populations and offer a deeper understanding of the unique properties of single Enzyme...

  • Digital concentration readout of single Enzyme Molecules using femtoliter arrays and Poisson statistics.
    Nano letters, 2006
    Co-Authors: David M. Rissin, David R. Walt
    Abstract:

    Methods for accurately quantifying the concentration of a particular analyte in solution are all based on ensemble responses in which many analyte Molecules give rise to the measured signal. In this paper, single Molecules of beta-galactosidase were monitored using a 1 mm diameter fiber optic bundle with 2.4 x 10(5) individually sealed, femtoliter microwell reactors. By observation of the buildup of fluorescent products from single Enzyme Molecule catalysis over the array of reaction vessels and by application of a Poisson statistical analysis, a digital concentration readout was obtained. This approach should prove useful for single Molecule enzymology and ultrasensitive bioassays. More generally, the ability to determine concentration by counting individual Molecules offers a new approach to analysis of dilute solutions.

Gerald R Smith - One of the best experts on this subject based on the ideXlab platform.

  • regulation of homologous recombination chi inactivates recbcd Enzyme by disassembly of the three subunits
    Genes & Development, 1999
    Co-Authors: Andrew F Taylor, Gerald R Smith
    Abstract:

    We report here an unusual mechanism for Enzyme regulation: the disassembly of all three subunits of RecBCD Enzyme after its interaction with a Chi recombination hot spot. The Enzyme, which is essential for the major pathway of recombination in Escherichia coli, acts on linear double-stranded DNA bearing a Chi site to produce single-stranded DNA substrates for strand exchange by RecA protein. We show that after reaction with DNA bearing Chi sites, RecBCD Enzyme is inactivated and the three subunits migrate as separate species during glycerol gradient ultracentrifugation or native gel electrophoresis. This Chi-mediated inactivation and disassembly of purified RecBCD Enzyme can account for the previously reported Chi-dependent loss of Chi activity in E. coli cells containing broken DNA. Our results support a model of recombination in which Chi regulates one RecBCD Enzyme Molecule to make a single recombinational exchange (‘one Enzyme-one exchange’ hypothesis).