Nine invariant residues among the sequences of all Ntn amidotransferases play key catalytic, substrate binding or structural roles.

[7][8] The free N terminus acts as a base to activate the nucleophile and protonate the leaving group in the hydrolytic reaction, in this case ammonia.

Rather, the primary function of the enzyme appears to be bringing the reactants together appropriately and preventing the wrong reaction, such as hydrolysis.

The ammonia released by the first reaction is then transferred to the active site of the phosphoribosyltransferase domain via a 20 Å channel, where it then binds to PRPP to form PRA.

[7][13][14] Inhibition occurs via a structural change in the enzyme where the flexible glutamine loop gets locked in an open position, preventing the binding of PRPP.

[7] Due to the chemical lability of PRA, which has a half-life of 38 seconds at pH 7.5 and 37 °C, researchers have suggested that the compound is channeled from Amidophosphoribosyltransferase to GAR synthetase in vivo.