Here the functional and structural importance of interactions involving the 240s loop of the catalytic chain for the stabilization of the T state of aspartate transcarbamoylase were tested by replacement of Lys-244 with Asn and Ala. For the K244A and K244N mutant enzymes, the aspartate concentration required to achieve half-maximal specific activity was reduced to 8.4 and 4.0 mm, respectively, as compared with 12.4 mM for the wild-type enzyme. Both mutant enzymes exhibited dramatic reductions in homotropic cooperativity and the ability of the heterotropic effectors to modulate activity. Small angle x-ray scattering studies showed that the unligated structure of the mutant enzymes, and the structure of the mutant enzymes ligated with N-phosphonacetyl-L-aspartate, were similar to that observed for the unligated and N-phosphonacetyl-L-aspartateligated wild-type enzyme. A saturating concentration of carbamoyl phosphate alone has little influence on the small angle x-ray scattering of the wild-type enzyme. However, carbamoyl phosphate was able to shift the structure of the two mutant enzymes dramatically toward R, establishing that the mutations had destabilized the T state of the enzyme. The x-ray crystal structure of K244N enzyme showed that numerous local T state stabilizing interactions involving 240s loop residues were lost. Furthermore, the structure established that the mutation induced additional alterations at the subunit interfaces, the active site, the relative position of the domains of the catalytic chains, and the allosteric domain of the regulatory chains. Most of these changes reflect motions toward the R state structure. However, the K244N mutation alone only changes local conformations of the enzyme to an R-like structure, without triggering the quaternary structural transition. These results suggest that loss of cooperativity and reduction in heterotropic effects is due to the dramatic destabilization of the T state of the enzyme by this mutation in the 240s loop of the catalytic chain.