Substrate specificity of aspartate transcarbamylase. Interaction of the enzyme with analogs of aspartate and succinate.
The ability of aspartate transcarbamylase from Escherichia coli to catalyze carbamylation of amino acids other than the natural substrate, L-aspartate, was examined. Cysteine, cysteate, cysteinesulfinate, and 3-nitroalanine showed kcat values at pH 7 of 0.16, 0.58, 5.2, and 62 s-1, respectively, while kcat with aspartate was 320 s-1. In a parallel study, competitive inhibition constants of 3-nitropropionate, 3-mercaptopropionate, 3-sulfopropionate, and 3-sulfinopropionate were found to be high, about 0.1 M, compared with that of succinate, 0.56 mM. Although cysteinesulfinate had low activity as a substrate, the pH dependences of kcat and kcat/Km in H2O and D2O observed with the compound closely paralleled those of aspartate. The results of these studies suggest that substrate specificity and reactivity are achieved in part by a strong, highly specific interaction of one or more active site residues with the beta-carboxylate of L-aspartate. Unlike the sigmoidal kinetics found with aspartate, saturation of native aspartate transcarbamylase by cysteine sulfinate showed a lack of cooperativity, even under conditions of activation of the reaction by ATP and inhibition by CTP. The cysteinesulfinate reaction was increased 9-fold by the bisubstrate analog N-phosphonacetyl-L-aspartate. These results were interpreted in terms of an inability of cysteinesulfinate to cause the allosteric conformational change promoted by aspartate.
D000596:Amino Acids / Q000378:metabolism; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018377:Neurotransmitter Agents; D013379:Substrate Specificity; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid
D000596:Amino Acids; D018377:Neurotransmitter Agents; D013386:Succinates; D001224:Aspartic Acid; D019802:Succinic Acid; D001221:Aspartate Carbamoyltransferase; D003545:Cysteine; C013461:cysteine sulfinic acid