The ATP-grasp fold is found in enzymes that catalyze the formation of an amide bond and occurs twice in carbamoyl phosphate synthetase. We have used site-directed mutagenesis to further define the relationship of these ATP folds to the ATP-grasp family and to probe for distinctions between the two ATP sites. Mutations at D265 and D810 severely diminished activity, consistent with consensus ATP-grasp roles of facilitating the transfer of the gamma-phosphate group of ATP. H262N was inactive whereas H807N, the corresponding mutation in the second ATP domain, exhibited robust activity, suggesting that these residues were not involved in the ATP-grasp function common to both domains. Mutations at I316 were somewhat catalytically impaired and were structurally unstable, consistent with a consensus role of interaction with the adenine and/or ribose moiety of ATP. L229G was too unstable to be purified and characterized. S228A showed essentially wild-type behavior.