Most bacteria and all archaea misacylate the tRNAs corresponding to Asn and Gln with Asp and Glu (Asp-tRNA(Asn) and Glu-tRNA(Gln)).The GatCAB enzyme of most bacteria converts misacylated Glu-tRNA(Gln) to Gln-tRNA(Gln) in order to enable the incorporation of glutamine during protein synthesis. The conversion process involves the intramolecular transfer of ammonia between two spatially separated active sites. This study presents a computational analysis of the two putative intramolecular tunnels that have been suggested to describe the ammonia transfer between the two active sites. Molecular dynamics simulations have been performed for wild-type GatCAB of S. aureus and its mutants: T175((A))V, K88((B))R, E125((B))D, and E125((B))Q. The two tunnels have been analyzed in terms of free energy of ammonia transfer along them. The probability of occurrence of each type of tunnel and the variation of the probability for wild-type GatCAB and its mutants is also discussed.