The amidotransferase family of enzymes: Molecular machines for the production and delivery of ammonia

The amidotransferase family of enzymes utilizes the ammonia derived from th e hydrolysis of glutamine for a subsequent chemical reaction catalyzed by t he same enzyme. The ammonia intermediate does not dissociate into solution during the chemical transformations. A well-characterized example of the st ructure and mechanism displayed by this class of enzymes is provided by car bamoyl phosphate synthetase (CPS). Carbamoyl phosphate synthetase is isolat ed from Escherichia coli as a heterodimeric protein. The smaller of the two subunits catalyzes the hydrolysis of glutamine to glutamate and ammonia. T he larger subunit catalyzes the formation of carbamoyl phosphate using 2 mo l of ATP, bicarbonate, and ammonia. Kinetic investigations have led to a pr oposed chemical mechanism for this enzyme that requires carboxy phosphate, ammonia, and carbamate as kinetically competent reaction intermediates. The three-dimensional X-ray crystal structure of CPS has localized the positio ns of three active sites. The nucleotide binding site within the N-terminal half of the large subunit is required for the phosphorylation of bicarbona te and subsequent formation of carbamate. The nucleotide binding site withi n the C-terminal domain of the large subunit catalyzes the phosphorylation of carbamate to the final product, carbamoyl phosphate. The three active si tes within the heterodimeric protein are separated from one another by abou t 45 Angstrom. The ammonia produced within the active site of the small sub unit is the substrate for reaction with the carboxy phosphate intermediate that is formed in the active site found within the N-terminal half of the l arge subunit of CPS. Since the ammonia does not dissociate from the protein prior to its reaction with carboxy phosphate, this intermediate must there fore diffuse through a molecular tunnel that connects these two sites with one another. Similarly, the carbamate intermediate, initially formed at the active site within the N-terminal half of the large subunit, is the substr ate for phosphorylation by the ATP bound to the active site located in the C-terminal half of the large subunit. A molecular passageway has been ident ified by crystallographic methods that apparently facilitates diffusion bet ween these two active sites within the large subunit of CPS. Synchronizatio n of the chemical transformations is controlled by structural perturbations among the three active sites. Molecular tunnels between distant active sit es have also been identified in tryptophan synthase and glutamine phosphori bosyl pyrophosphate amidotransferase and are likely architectural features in an expanding list of enzymes.