Cooperative Interactions in Aspartate Transcarbamoylase. 1. Hybrids Composed of Native and Chemically Inactivated Catalytic Polypeptide Chains

Gibbons, I. R.; Yang, Ying; Schachman, H. K.

doi:10.1073/pnas.71.11.4452

Cited by 34 publications

(37 citation statements)

References 21 publications

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“…introduced at chemically defined amino acid side-chains, not necessarily in the active site. Reversible chemical modification to produce hybrid forms of oligomeric enzymes promises to be a welcome addition to the techniques for studying subunit interactions [86,87]. However, the relevance to the present article of such experiments lies not so much in the value of the results they have produced but in the fact that they would not have been conceived of without the development of our general knowledge of the protein chemistry of enzymes outlined above.…”

Section: Topography and Three-dimensional Structurementioning

confidence: 77%

The Protein chemistry of enzymes

Perham

1976

FEBS Letters

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Section: Topography and Three-dimensional Structurementioning

confidence: 77%

The Protein chemistry of enzymes

Perham

1976

FEBS Letters

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“…Another similarity between the two hybrid holoenzymes is the observed decrease in cooperativity relative to the wild-type enzyme. This reduction is to be expected as the number of active sites has effectively been reduced by 50% and is reminiscent of the earlier active/inactive hybrids (27).…”

Section: Small Angle X-ray Scattering Of the (R105a-c)(at-c)rmentioning

confidence: 99%

Importance of Domain Closure for the Catalysis and Regulation ofEscherichia coli Aspartate Transcarbamoylase

Macol¹,

Tsuruta²,

Kantrowitz³

2002

Journal of Biological Chemistry

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Two hybrid versions of Escherichia coli aspartate transcarbamoylase were studied to determine the influence of domain closure on the homotropic and heterotropic properties of the enzyme. Each hybrid holoenzyme had one wild-type and one inactive catalytic subunit. In the first case the inactive catalytic subunit had Arg-54 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 17,000-fold reduction in activity, no loss in substrate affinity, and an R state structurally identical to that of the wild-type enzyme. In the second case, the inactive catalytic subunit had Arg-105 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 1,100-fold reduction in activity, substantial loss in substrate affinity, and loss of the ability to be converted to the R state. Thus, the R54A substitution results in a holoenzyme that can undergo closure of the catalytic chain domains to form the high activity, high affinity active site and to undergo the allosteric transition, whereas the R105A substitution results in a holoenzyme that can neither undergo domain closure nor the allosteric transition. The hybrid holoenzyme with one wild-type and one R54A catalytic subunit exhibited the same maximal velocity per active site as the wild-type holoenzyme, reduced cooperativity, and normal heterotropic interactions. The hybrid with one wild-type and one R105A catalytic subunit exhibited significantly reduced maximal velocity per active site as compared with the wild-type holoenzyme, reduced cooperativity, and substantially reduced heterotropic interactions. Small angle x-ray scattered was used to verify that the R105A-containing hybrid could attain an R state structure. These results indicate the global nature of the conformational changes associated with the allosteric transition in the enzyme. If one catalytic subunit cannot undergo domain closure to create the active sites, then the entire molecule cannot attain the high activity, high activity R state.The homotropic cooperativity in Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) is directly related to the ability of the substrates to induce a structural and functional transition of the enzyme from a low activity low affinity T state to a high activity, high affinity R state (1). Structurally the conversion of the enzyme from the T to the R state involves an elongation of the enzyme by 11 Å along the molecular 3-fold axis along with a twist of the two catalytic subunits of about 5°in opposite directions (2). This structural transition also results in a simultaneous rotation of each regulatory dimer about its own 2-fold axis. In addition to these quaternary structural changes, the T to R transition involves structural alterations on the tertiary level. For example, during the T to R transition each catalytic chain undergoes a domain closure in which the aspartate domain moves 3 Å toward the carbamoyl phosphate domain, resulting in a major reorganization of the interdomain interface. In addition to this ...

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“…The acylated derivatives were stable for many hours at pH 8 and deacylation was achieved overnight at pH 6. With this procedure coupled with pyridoxylation of the C trimer (which had been shown by Stark and his colleagues to inactivate the enzyme), Gibbons, Yang, and I demonstrated that an ATCase hybrid containing one active and one chemically inactivated C trimer was allosteric (18). Analogous procedures were used to make an ATCase hybrid with one unmodified and two pyridoxylated c chains in each trimer.…”

Section: Living With Hybrid Atcase Moleculesmentioning

confidence: 99%

Still Looking for the Ivory Tower

Schachman

2000

Annu. Rev. Biochem.

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Following graduate training, which was disrupted by my changing schools and serving in the Navy in World War II, I arrived in Berkeley in 1948 as an instructor in the Biochemistry Department. Despite numerous academic reorganizations and a host of struggles over the University-imposed Loyalty Oath, dismissal of a faculty member because of political affiliations, free speech for students, and my resistance to mandatory retirement, I survived with the help of great graduate students, postdoctoral fellows, undergraduates, superb research assistants, and a supportive wife. Studies on structure of tobacco mosaic virus led to our investigating an ultracentrifuge anomaly and the construction of a synthetic boundary cell. In turn, this resulted in about 15 years of research on the ultracentrifuge and its application to the study of biological macromolecules. Among the latter, the discovery of large ribonucleoprotein complexes, now known as ribosomes, and chromatophores in photosynthetic microorganisms attracted the most attention. But it was the development of the photoelectric absorption optical system and the incorporation of the Rayleigh interferometer onto the ultracentrifuge that had the greatest impact on our further research. These tools, when applied to our initial research on E. coli aspartate transcarbamoylase (ATCase), led to the discovery of distinct subunits for catalysis and regulation and the global conformational change in the enzyme associated with its role in regulation. For almost 35 years we have been using the techniques of protein chemistry and molecular biology in studies of structural and conformational changes in the enzyme, the genes encoding the different polypeptides, subunit interactions, and assembly of the enzyme from six catalytic and six regulatory chains. Hybrids constructed from inactive mutants were used to demonstrate shared active sites requiring the joint participation of amino acid residues from adjoining polypeptide chains. ATCase is still being studied as a model for understanding allostery as a regulatory mechanism. Circularly permuted polypeptide chains are being used to study the folding and assembly pathways, and the recently determined crystal structure of the active nonallosteric catalytic subunit has led to new questions regarding the activated form of ATCase.

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Cooperative Interactions in Aspartate Transcarbamoylase. 1. Hybrids Composed of Native and Chemically Inactivated Catalytic Polypeptide Chains

Cited by 34 publications

References 21 publications

The Protein chemistry of enzymes

The Protein chemistry of enzymes

Importance of Domain Closure for the Catalysis and Regulation ofEscherichia coli Aspartate Transcarbamoylase

Still Looking for the Ivory Tower

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