Multifunctional enzymes catalyzing successive reactions have evolved several mechanisms for the transport of intermediates between active sites. One mechanism, substrate channeling, allows the transport of the intermediate without releasing it into the solvent. Members of the glutamine amidotransferase (GAT) family often utilize substrate channeling for the transport of intermediates. GAT enzymes hydrolyze glutamine to ammonia, which is transported to an acceptor domain preventing wasteful hydrolysis of glutamine and increasing the efficiency of the reaction. Many GAT enzymes utilize molecular tunnels to shuttle ammonia between active sites. Often GAT enzymes synchronize the active site through conformational changes that occur during catalysis.

Glutamine-dependent NAD synthetases are GAT enzymes and catalyze the last step in the biosynthesis of NAD, utilizing nicotinic acid adenine dinucleotide (NaAD), ATP and glutamine. Steady-state kinetic characterizations and stoichiometric analysis of NAD synthetase from Mycobacterium tuberculosis (NAD synthetaseTB) revealed a substrate channeling mechanism for ammonia transport and tight coordination of the active sites resulting in an enzyme that is highly efficient in the use of glutamine. The crystal structure of NAD synthetaseTB has revealed a 40 Å tunnel that connects the active sites and is postulated to play a role in the synchronized activities. Several regions of the enzyme were identified that may be important for regulation, such as the YRE loop which contacts the glutamine active site and key regions of the tunnel. Mutations of tunnel residues, such as D656A, show that interruption of important interactions can result in compromise in transfer of ammonia or active site communication.

Phylogenetic analysis revealed that glutamine-dependent NAD synthetases have different levels of regulation. Three groups of enzymes were identified represented by NAD synthetase from M. tuberculosis, S. cerevisiae (NAD synthetaseYeast) and Thermotoga maritima (NAD synthetaseTM). Steady-state kinetic characterizations and stoichiometric analysis of NAD synthetaseTM has revealed a compromised coordination of the active sites compared to the highly synchronized NAD synthetaseTB and the moderate synchronization of NAD synthetaseYeast. Sequence alignment of these groups has allowed identification of residues that line the tunnel that may be responsible for the differences observed in active site coordination and are, therefore, important for active site communication.