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Abstract
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.
Highlights
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]
The enzyme was crystallized with 10 times as much PALA as the wild-type, the structure of the R105A enzyme was in the T state with no PALA bound at the active site [7]
In the work reported here, two hybrid versions of aspartate transcarbamoylase were constructed and characterized to understand more fully how substrate binding and the subsequent tertiary movements in one catalytic subunit affect the other catalytic subunit in the holoenzyme, and how these tertiary movements are related to the global allosteric transition
Summary
Materials—Agarose, ATP, CTP, L-aspartate, N-carbamoyl-L-aspartate, potassium dihydrogen phosphate, and uracil were obtained from Sigma. Ion-exchange chromatography using Q-Sepharose Fast Flow resin was used to initially purify the regulatory subunit [16]. After the sample was loaded, the column was washed with 5 column volumes of 50 mM Tris acetate buffer, pH 8.3, 2 mM 2-mercaptoethanol, 0.1 mM zinc acetate This was followed by a gradient (total volume of 10 column volumes) of 0 –50% 50 mM Tris acetate buffer, pH 8.3, 2 mM 2-mercaptoethanol, 0.1 mM zinc acetate, 1.0 M NaCl. After concentration of the appropriate fractions, the purity of the regulatory subunit was checked by SDS-PAGE [13] and nondenaturing PAGE [14, 15].
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