Substitutions in the aspartate transcarbamoylase domain of hamster CAD disrupt oligomeric structure.
Aspartate transcarbamoylase (ATCase; EC 2.1.3.2) is one of three enzymatic domains of CAD, a protein whose native structure is usually a hexamer of identical subunits. Alanine substitutions for the ATCase residues Asp-90 and Arg-269 were generated in a bicistronic vector that encodes a 6-histidine-tagged hamster CAD. Stably transfected mammalian cells expressing high levels of CAD were easily isolated and CAD purification was simplified over previous procedures. The substitutions reduce the ATCase V(max) of the altered CADs by 11-fold and 46-fold, respectively, as well as affect the enzyme's affinity for aspartate. At 25 mM Mg(2+), these substitutions cause the oligomeric CAD to dissociate into monomers. Under the same dissociating conditions, incubating the altered CAD with the ATCase substrate carbamoyl phosphate or the bisubstrate analogue N-phosphonacetyl-L-aspartate unexpectedly leads to the reformation of hexamers. Incubation with the other ATCase substrate, aspartate, has no effect. These results demonstrate that the ATCase domain is central to hexamer formation in CAD and suggest that the ATCase reaction mechanism is ordered in the same manner as the Escherichia coli ATCase. Finally, the data indicate that the binding of carbamoyl phosphate induces conformational changes that enhance the interaction of CAD subunits.
Qiu
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Department of Microbiology, Albert B. Chandler Medical Center, University of Kentucky, Lexington, KY 40536-0084, USA.
Davidson
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D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000235:genetics*; D001224:Aspartic Acid / Q000031:analogs & derivatives; D016466:CHO Cells; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry / Q000235:genetics*; D002221:Carbamyl Phosphate / Q000378:metabolism; D006224:Cricetinae; D004080:Dihydroorotase / Q000737:chemistry / Q000235:genetics*; D004686:Encephalomyocarditis virus / Q000235:genetics; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives; D010957:Plasmids; D011487:Protein Conformation; D014162:Transfection
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D001224:Aspartic Acid; D002221:Carbamyl Phosphate; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D010746:Phosphonoacetic Acid