Aspartate-90 and arginine-269 of hamster aspartate transcarbamylase affect the oligomeric state of a chimaeric protein with an Escherichia coli maltose-binding domain.
Residues Asp-90 and Arg-269 of Escherichia coli aspartate transcarbamylase seem to interact at the interface of adjacent catalytic subunits. Alanine substitutions at the analogous positions in the hamster aspartate transcarbamylase of a chimaeric protein carrying an E. coli maltose-binding domain lead to changes in both the kinetics of the enzyme and the quaternary structure of the protein. The Vmax for the Asp-90-->Ala and Arg-269-->Ala substitutions is decreased to 1/21 and 1/50 respectively, the [S]0.5 for aspartate is increased 540-fold and 826-fold respectively, and the [S]0.5 for carbamoyl phosphate is increased 60-fold for both. These substitutions decrease the oligomeric size of the protein. Whereas the native chimaeric protein behaves as a pentamer, the Asp-90 variant is a trimer and the Arg-269 variant is a dimer. The altered enzymes also exhibit marked decreases in thermal stability and are inactivated at much lower concentrations of urea than is the unaltered enzyme. Taken together, these results are consistent with the hypothesis that both Asp-90 and Arg-269 have a role in the enzymic function and structural integrity of hamster aspartate transcarbamylase.
Qiu
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Department of Microbiology and Immunology, Albert B. Chandler Medical Center, University of Kentucky, Lexington, KY 40536-0084, USA.
Davidson
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D018528:ATP-Binding Cassette Transporters; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D007700:Kinetics; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014508:Urea / Q000737:chemistry
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D001224:Aspartic Acid; D014508:Urea; D001120:Arginine; D001221:Aspartate Carbamoyltransferase