Structure of Carbamoyl Phosphate Synthetase:  A Journey of 96 Å from Substrate to Product^,

Abstract

Carbamoyl phosphate synthetase catalyzes the production of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of MgATP. As isolated from Escherichia coli, the enzyme has a total molecular weight of approximately 160K and consists of two polypeptide chains referred to as the large and small subunits. Here we describe the X-ray crystal structure of this enzyme determined to 2.8 A resolution in the presence of ADP, Mn2+, phosphate, and ornithine. The small subunit is distinctly bilobal with the active site residues located in the interface formed by the NH2- and COOH-terminal domains. Interestingly, the structure of the COOH-terminal half is similar to that observed in the trpG-type amidotransferase family. The large subunit can be envisioned as two halves referred to as the carboxyphosphate and carbamoyl phosphate synthetic components. Each component contains four distinct domains. Strikingly, the two halves of the large subunit are related by a nearly exact 2-fold rotational axis, thus suggesting that this polypeptide chain evolved from a homodimeric precursor. The molecular motifs of the first three domains observed in each synthetic component are similar to those observed in biotin carboxylase. A linear distance of approximately 80 A separates the binding sites for the hydrolysis of glutamine in the small subunit and the ATP-dependent phosphorylations of bicarbonate and carbamate in the large subunit. The reactive and unstable enzyme intermediates must therefore be sequentially channeled from one active site to the next through the interior of the protein.

Localization of the site for the nucleotide effectors ofEscherichia colicarbamoyl phosphate synthetase using site‐directed mutagenesis

“…Based on the photoa¤nity labeling by UMP and IMP at these residues and on the e¡ects of replacing His-995 by alanine on the binding of the two nucleotide e¡ectors [6], we con¢rmed that these e¡ectors bind at overlapping sites, and we proposed the involvement of speci¢c sequence regions around Lys-993 in the formation of the binding site for the nucleotide e¡ectors [5,6]. Recently the three-dimensional structure of E. coli CPS was determined and a P i ion was localized binding to the crystal in the region assigned by us to the e¡ector nucleotide site [7]. We have used the position of this P i and the sites of photoa¤nity labeling by UMP and IMP to model the bound e¡ector nucleotides [6] (Fig.…”

“…2), represented 16.5% of the protein in the extract for wild-type CPS and 8.8%, 6% and 13% for the CPSs with the mutations Ser-948-Ala, Lys-954-Ala and Thr-974-Ala, respectively. Enzyme activity (assayed by coupling with ornithine transcarbamylase as indicated in Section 2, in the presence of 10 mM ATP, 20 mM MgCl P and 4 mM ornithine) was, in Wmol/min/mg protein in the extract, 0.79, 0.13, 0.07 and 1.9 [7]. IMP structure was taken from the library of heterocompounds of Uppsala University (http://alpha2.bmc.uu.se/hicup/).…”

Gain of Glutaminase Function in Mutants of the Ammonia-specific Frog Carbamoyl Phosphate Synthetase

“…eCPS, bind and cleave glutamine at a glutamine amidotransferase (GAT) domain and channel the resulting free ammonia, sequestered within the enzyme, to a synthetase (SYN) domain where all other ligands are bound and all other reactions take place (7,8). Ammonia can substitute for glutamine in eCPS, but with a much higher K m (111 versus 0.17 mM; Ref.…”

Resolving the Fluorescence Response of Escherichia coli Carbamoyl Phosphate Synthetase: Mapping Intra- and Intersubunit Conformational Changes

“…for the two ligands have since been verified via crystal structures (3,18). A third molecule, IMP, is competitive with UMP and was originally implicated as an allosteric activator of E. coli CPS (2).…”