Production and Crystallization of Aspartate Transcarbamylase

Shepherdson, Margaret; Pardee, Arthur B.

doi:10.1016/s0021-9258(20)81343-x

Cited by 85 publications

(12 citation statements)

References 11 publications

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“…Discovered in the 1960s, ATCase was found to control the rate of pyrimidine biosynthesis via feedback inhibition. 160 This initial discovery also identified several ligand-binding sites distinct from the active site and determined that thermodynamic interactions between the sites could be uncoupled. 160,161 X-ray crystallography studies identified twofold and threefold symmetry axes within the enzyme, giving a dodecameric Ct 6 Rg 6 structure containing two catalytic (Ct) trimers and three regulatory (Rg, to distinguish from "R" conformational state) dimers.…”

Section: Structure and Functionmentioning

confidence: 99%

“…160 This initial discovery also identified several ligand-binding sites distinct from the active site and determined that thermodynamic interactions between the sites could be uncoupled. 160,161 X-ray crystallography studies identified twofold and threefold symmetry axes within the enzyme, giving a dodecameric Ct 6 Rg 6 structure containing two catalytic (Ct) trimers and three regulatory (Rg, to distinguish from "R" conformational state) dimers. 163−165 Like hemoglobin, its allosteric behavior conforms to the MWC model, with the apoenzyme favoring the inactive T-state nearly 250:1 based on kinetic studies.…”

Section: Structure and Functionmentioning

confidence: 99%

“…The reaction proceeds through a tetrahedral intermediate and can be inhibited by the tight-binding bisubstrate analogue N -(phosphonoacetyl)- l -aspartate (PALA) that mimics the reaction intermediate structure. Discovered in the 1960s, ATCase was found to control the rate of pyrimidine biosynthesis via feedback inhibition . This initial discovery also identified several ligand-binding sites distinct from the active site and determined that thermodynamic interactions between the sites could be uncoupled. , …”

Section: Aspartate Transcarbamoylasementioning

confidence: 99%

“…Discovered in the 1960s, ATCase was found to control the rate of pyrimidine biosynthesis via feedback inhibition . This initial discovery also identified several ligand-binding sites distinct from the active site and determined that thermodynamic interactions between the sites could be uncoupled. , …”

Section: Aspartate Transcarbamoylasementioning

confidence: 99%

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Solution NMR Spectroscopy for the Study of Enzyme Allostery

2016

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Allostery is a ubiquitous biological regulatory process in which distant binding sites within a protein or enzyme are functionally and thermodynamically coupled. Allosteric interactions play essential roles in many enzymological mechanisms, often facilitating formation of enzyme-substrate complexes and/or product release. Thus, elucidating the forces that drive allostery is critical to understanding the complex transformations of biomolecules. Currently, a number of models exist to describe allosteric behavior, taking into account energetics as well as conformational rearrangements and fluctuations. In the following review, we discuss the use of solution NMR techniques designed to probe allosteric mechanisms in enzymes. NMR spectroscopy is unequaled in its ability to detect structural and dynamical changes in biomolecules, and the case studies presented herein demonstrate the range of insights to be gained from this valuable method. We also provide a detailed technical discussion of several specialized NMR experiments that are ideally suited for the study of enzymatic allostery.

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Section: Structure and Functionmentioning

confidence: 99%

Section: Structure and Functionmentioning

confidence: 99%

Section: Aspartate Transcarbamoylasementioning

confidence: 99%

Section: Aspartate Transcarbamoylasementioning

confidence: 99%

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Solution NMR Spectroscopy for the Study of Enzyme Allostery

2016

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“…Margaret Shepherdson worked briefly in Art's laboratory to purify the enzyme after derepressing the pathway in a pyrimidine auxotroph to raise the protein's level to several percent of the cell's total (Shepherdson & Pardee 1960), and then she returned to England, bequeathing me a tube of purified ATCase. I undertook a kinetic study, measuring enzyme Enzymatic activity (arb.…”

Section: Metabolic Regulationmentioning

confidence: 99%

Untitled

2010

Annu. Rev. Cell Dev. Biol.

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In the 1950s, cellular regulatory mechanisms were newly recognized; with Arthur Pardee I investigated the initial enzyme of pyrimidine biosynthesis, which he discovered is controlled by feedback inhibition. The protein proved unusual in having separate but interacting sites for substrates and regulators. Howard Schachman and I dissociated the protein into different subunits, one binding regulators and one substrates. The enzyme became an early prime example of allostery. In developmental biology I studied the egg of the frog, Xenopus laevis, characterizing early processes of axis formation. My excellent students and I described cortical rotation, a 30 • movement of the egg's cortex over tracks of parallel microtubules anchored to the underlying cytoplasmic core, and we perturbed it to alter Spemann's organizer and effect spectacular phenotypes. The entire sequence of events has been elucidated by others at the molecular level, making Xenopus a prime example of vertebrate axis formation. Marc Kirschner, Christopher Lowe, and I then compared hemichordate (half-chordate) and chordate early development. Despite anatomical-physiological differences, these groups share numerous steps of axis formation, ones that were probably already in use in their pre-Cambrian ancestor. I've thoroughly enjoyed exploring these areas during a 50-year period of great advances in biological sciences by the worldwide research community.

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Aspartate Transcarbamylase from Escherichia Coli: Activity and Regulation

Lipscomb¹

1994

Advances in Enzymology - And Related Areas of Molecular Biology

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Production and Crystallization of Aspartate Transcarbamylase

Cited by 85 publications

References 11 publications

Solution NMR Spectroscopy for the Study of Enzyme Allostery

Solution NMR Spectroscopy for the Study of Enzyme Allostery

Untitled

Aspartate Transcarbamylase from Escherichia Coli: Activity and Regulation

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