ABSTRACT
Homotropic cooperativity in Escherichia coli aspartate transcarbamoylase results from the substrate-induced transition from the T to the R state. These two alternate states are stabilized by a series of interdomain and intersubunit interactions. The salt link between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain is only observed in the T state. When Asp-236 is replaced by alanine the resulting enzyme exhibits full activity, enhanced affinity for aspartate, no cooperativity, and no heterotropic interactions. These characteristics are consistent with an enzyme locked in the functional R state. Using small angle x-ray scattering, the structural consequences of the D236A mutant were characterized. The unliganded D236A holoenzyme appears to be in a new structural state that is neither T, R, nor a mixture of T and R states. The structure of the native D236A holoenzyme is similar to that previously reported for another mutant holoenzyme (E239Q) that also lacks intersubunit interactions. A hybrid version of aspartate transcarbamoylase in which one catalytic subunit was wild-type and the other had the D236A mutation was also investigated. The hybrid holoenzyme, with three of the six possible interactions involving Asp-236, exhibited homotropic cooperativity, and heterotropic interactions consistent with an enzyme with both T and R functional states. Small angle x-ray scattering analysis of the unligated hybrid indicated that the enzyme was in a new structural state more similar to the T than to the R state of the wild-type enzyme. These data suggest that three of the six intersubunit interactions involving D236A are sufficient to stabilize a T-like state of the enzyme and allow for an allosteric transition.
Subject(s)
Aspartate Carbamoyltransferase/chemistry , Aspartate Carbamoyltransferase/isolation & purification , Aspartic Acid/analogs & derivatives , Escherichia coli/enzymology , Phosphonoacetic Acid/analogs & derivatives , Adenosine Triphosphate/metabolism , Alanine/chemistry , Allosteric Site , Aspartic Acid/chemistry , Aspartic Acid/pharmacology , Catalytic Domain , Chromatography, Ion Exchange , Cytidine Triphosphate/metabolism , Enzyme Inhibitors/pharmacology , Ligands , Lysine/chemistry , Models, Molecular , Mutation , Phosphonoacetic Acid/pharmacology , Protein Binding , Protein Structure, Tertiary , Scattering, Radiation , X-RaysABSTRACT
This study shows that vascular smooth muscle cells express significantly higher levels of gamma interferon-inducible indoleamine 2,3-dioxygenase (IDO) activity than endothelium or mononuclear cells. Since IDO activity is linked to persistent Chlamydophila pneumoniae infection, our results suggest that smooth muscle cells may be an important reservoir of that organism in atherosclerosis.
