Role of the Hinge Loop Linking the N- and C-Terminal Domains of the Amidotransferase Subunit of Carbamoyl Phosphate Synthetase

Abstract

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme consists of a large synthetase subunit and a small amidotransferase subunit. The small subunit is structurally bilobal. The N-terminal domain is unique compared to the sequences of other known proteins. The C-terminal domain, which contains the direct catalytic residues for the amidotransferase activity of CPS, is homologous to other members of the Triad glutamine amidotransferases. The two domains are linked by a hinge-like loop, which contains a type II β turn. The role of this loop in the hydrolysis of glutamine and the formation of carbamoyl phosphate was probed by site-directed mutagenesis. Based upon the observed kinetic properties of the mutants, the modifications to the small subunit can be separated into two groups. The first group consists of G152I, G155I, and Δ155. Attempts to disrupt the turn conformation were made by the deletion of Gly-155 or substitution of the two glycine residues with isoleucine. However, these mutations only have minor effects on the kinetic properties of the enzyme. The second group includes L153W, L153G/N154G, and a ternary complex consisting of the intact large subunit plus the separate N- and C-terminal domains of the small subunit. Although the ability to synthesize carbamoyl phosphate is retained in these enzymes, the hydrolysis of glutamine is partially uncoupled from the synthetase reaction. It is concluded that the hinge loop, but not the type-II turn structure of the loop per se, is important for maintaining the proper interface interactions between the two subunits and the catalytic coupling of the partial reactions occurring within the separate subunits of CPS.