It is generally accepted that naturally existing functional domains can serve as building blocks for complex protein structures, and that novel functions can arise from assembly of different combinations of these functional domains. To inform our understanding of protein evolution and explore the modular nature of protein structure, two model enzymes were chosen for study, purT-encoded glycinamide ribonucleotide formyltransferase (PurT) and purK-encoded N(5)-carboxylaminoimidazole ribonucleotide synthetase (PurK). Both enzymes are found in the de novo purine biosynthetic pathway of Escherichia coli. In spite of their low sequence identity, PurT and PurK share significant similarity in terms of tertiary structure, active site organization, and reaction mechanism. Their characteristic three domain structures categorize both PurT and PurK as members of the ATP-grasp protein superfamily. In this study, we investigate the exchangeability of individual protein domains between these two enzymes and the in vivo and in vitro functional properties of the resulting hybrids. Six domain-swapped hybrids were unable to catalyze full wild-type reactions, but each hybrid protein could catalyze partial reactions. Notably, an additional loop replacement in one of the domain-swapped hybrid proteins was able to restore near wild-type PurK activity. Therefore, in this model system, domain-swapped proteins retained the ability to catalyze partial reactions, but further modifications were required to efficiently couple the reaction intermediates and achieve catalysis of the full reaction. Implications for understanding the role of domain swapping in protein evolution are discussed.