Kinetics of the allosteric transition of aspartate transcarbamylase chemical quench studies

Kihara, Hiroshi; Barman, Thomas E.; Jones, Peter; Moody, Michael F.

doi:10.1016/0022-2836(84)90175-x

Cited by 6 publications

(1 citation statement)

References 29 publications

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“…Taking advantage of the high flux from synchrotron radiation X-ray beams, time-resolved measurements were performed using the SAXS beamline BL15A at the Photon Factory, a second-generation machine (Wakabayashi & Amemiya, 1991), and a stopped-flow apparatus specifically designed for SAXS measurements and fast mixing of viscous solutions (Tsuruta et al 1989). Indeed, the expected rate of the conformational change [typical times of the order of 10 ms at 4 xC (Kihara et al 1984)], left no hope of following the transition at room temperature. Experiments were thus performed in 20-30 % ethylene glycol at x5 to x10 xC, conditions in which the enzyme was shown to be still cooperative and sensitive to allosteric effectors.…”

Section: Structural Changes and Catalytic Activity Of The Allosteric mentioning

confidence: 99%

Small-angle scattering: a view on the properties, structures and structural changes of biological macromolecules in solution

Koch

Vachette

Svergun

2003

Quart. Rev. Biophys.

510

481

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A self-contained presentation of the main concepts and methods for interpretation of X-ray and neutron-scattering patterns of biological macromolecules in solution, including a reminder of the basics of X-ray and neutron scattering and a brief overview of relevant aspects of modern instrumentation, is given. For monodisperse solutions the experimental data yield the scattering intensity of the macromolecules, which depends on the contrast between the solvent and the particles as well as on their shape and internal scattering density fluctuations, and the structure factor, which is related to the interactions between macromolecules. After a brief analysis of the information content of the scattering intensity, the two main approaches for modelling the shape and/or structure of macromolecules and the global minimization schemes used in the calculations are presented. The first approach is based, in its more advanced version, on the spherical harmonics approximation and relies on few parameters, whereas the second one uses bead models with thousands of parameters. Extensions of bead modelling can be used to model domain structure and missing parts in high-resolution structures. Methods for computing the scattering patterns from atomic models including the contribution of the hydration shell are discussed and examples are given, which also illustrate that significant differences sometimes exist between crystal and solution structures. These differences are in some cases explainable in terms of rigid-body motions of parts of the structures. Results of two extensive studies--on ribosomes and on the allosteric protein aspartate transcarbamoylase--illustrate the application of the various methods. The unique bridge between equilibrium structures and thermodynamic or kinetic aspects provided by scattering techniques is illustrated by modelling of intermolecular interactions, including crystallization, based on an analysis of the structure factor and recent time-resolved work on assembly and protein folding.

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Section: Structural Changes and Catalytic Activity Of The Allosteric mentioning

confidence: 99%

Small-angle scattering: a view on the properties, structures and structural changes of biological macromolecules in solution

Koch

Vachette

Svergun

2003

Quart. Rev. Biophys.

510

481

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The Rapid‐Flow‐Quench Method in the Study of Fast Reactions in Biochemistry: Extension to Subzero Conditions

Barman

Travers

1985

Methods of Biochemical Analysis

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The identification of chemical intermediates in enzyme catalysis by the rapid quench-flow technique

Barman

Bellamy

Gutfreund

et al. 2006

Cell. Mol. Life Sci.

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Traditionally, enzyme transient kinetics have been studied by the stopped-flow and rapid quench-flow (QF) methods. Whereas stopped-flow is the more convenient, it suffers from two weaknesses: optically silent systems cannot be studied, and when there is a signal it cannot always be assigned to a particular step in the reaction pathway. QF is a chemical sampling method; reaction mixtures are aged for a few milliseconds or longer, 'stopped' by a quenching agent and the product or the intermediate is measured by a specific analytical method. Here we show that by exploiting the array of current analytical methods and different quenching agents, the QF method is a key technique for identifying, and for characterising kinetically, intermediates in enzyme reaction pathways and for determining the order by which bonds are formed or cleaved by enzymes acting on polymer substrates such as DNA.

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Kinetics of the allosteric transition of aspartate transcarbamylase chemical quench studies

Cited by 6 publications

References 29 publications

Small-angle scattering: a view on the properties, structures and structural changes of biological macromolecules in solution

Small-angle scattering: a view on the properties, structures and structural changes of biological macromolecules in solution

The Rapid‐Flow‐Quench Method in the Study of Fast Reactions in Biochemistry: Extension to Subzero Conditions

The identification of chemical intermediates in enzyme catalysis by the rapid quench-flow technique

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