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

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.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 ...