STRUCTURE OF CARBAMOYL-PHOSPHATE SYNTHETASE - A JOURNEY OF 96 ANGSTROM FROM SUBSTRATE TO PRODUCT

Carbamoyl phosphate synthetase catalyzes the production of carbamoyl p hosphate from bicarbonate, glutamine, and two molecules of MgATP. As i solated from Escherichia coli, the enzyme has a total molecular weight of similar to 160K and consists of two polypeptide chains referred to as the large and small subunits. Here we describe the X-ray crystal s tructure of this enzyme determined to 2.8 Angstrom resolution in the p resence of ADP, Mn2+, phosphate, and ornithine. The small subunit is d istinctly bilobal with the active site residues located in the interfa ce formed by the NH2- and COOH-terminal domains. Interestingly, the st ructure of the COOH-terminal half is similar to that observed in the t rpG-type amidotransferase family. The large subunit can be envisioned as two halves referred to as the carboxyphosphate and carbamoyl phosph ate synthetic components, Each component contains four distinct domain s. Strikingly, the two halves of the large subunit are related by a ne arly exact 2-fold rotational axis, thus suggesting that this polypepti de chain evolved from a homodimeric precursor. The molecular motifs of the first three domains observed in each synthetic component are simi lar to those observed in biotin carboxylase. A linear distance of simi lar to 80 Angstrom separates the binding sites for the hydrolysis of g lutamine in the small subunit and the ATP-dependent phosphorylations o f bicarbonate and carbamate in the large subunit. The reactive and uns table enzyme intermediates must therefore be sequentially channeled fr om one active site to the next through the interior of the protein.