The attachment of AMP to the ureido (urea-like) group on citrulline activates the carbonyl center for subsequent nucleophilic attack.

[7] X-ray crystal structures have been generated for argininosuccinate synthetase from Thermus thermophilus, E. coli, Thermotoga maritime, and Homo sapiens.

In T. thermophilus, the ureido group of citrulline appears to be repositioned during nucleophilic attack to attain sufficient proximity to the α-phosphate of ATP.

[8] An argininosuccinate synthetase structure with a bound ATP in the active site has not been attained, although modeling suggests that the distance between ATP and the ureido group of citrulline is smaller in human argininosuccinate synthetase than in the E. coli variety, so it is likely that a much smaller conformational change is necessary for catalysis.

[6]{[9] Genetic defects that cause incorrect localization of argininosuccinate synthetase to the outer mitochondrial membrane cause type II citrullinemia.

[9] It is thought that regulation of argininosuccinate synthetase activity in arginine synthesis occurs primarily at the transcriptional level in response to glucocorticoids, cAMP, glucagon, and insulin.

In this role, argininosuccinate synthetase activity is regulated largely by inflammatory cellular signal molecules such as cytokines.

[6] In endothelial cells, it has been shown that ASS expression is increased by laminar shear stress due to pulsative blood flow.

A few mutations lead to the production of an abnormally short enzyme that cannot effectively play its role in the urea cycle.

An accumulation of ammonia during the first few days of life leads to poor feeding, vomiting, seizures, and the other signs and symptoms of type I citrullinemia.