Carbamoyl-phosphate Synthetase

Abstract

Carbamoyl-phosphate synthetase catalyzes the production of carbamoyl phosphate through a reaction mechanism requiring one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli is composed of two polypeptide chains. The smaller of these belongs to the Class I amidotransferase superfamily and contains all of the necessary amino acid side chains required for the hydrolysis of glutamine to glutamate and ammonia. Two homologous domains from the larger subunit adopt conformations that are characteristic for members of the ATP-grasp superfamily. Each of these ATP-grasp domains contains an active site responsible for binding one molecule of MgATP. High resolution x-ray crystallographic analyses have shown that, remarkably, the three active sites in the E. coli enzyme are connected by a molecular tunnel of ϳ100 Å in total length. Here we describe the high resolution x-ray crystallographic structure of the G359F (small subunit) mutant protein of carbamoyl phosphate synthetase. This residue was initially targeted for study because it resides within the interior wall of the molecular tunnel leading from the active site of the small subunit to the first active site of the large subunit. It was anticipated that a mutation to the larger residue would "clog" the ammonia tunnel and impede the delivery of ammonia from its site of production to the site of utilization. In fact, the G359F substitution resulted in a complete change in the conformation of the loop delineated by Glu-355 to Ala-364, thereby providing an "escape" route for the ammonia intermediate directly to the bulk solvent. The substitution also effected the disposition of several key catalytic amino acid side chains in the small subunit active site.

Next-Generation Annotation of Prokaryotic Genomes with EuGene-P: Application to Sinorhizobium meliloti 2011

“…This insertion, located between SMc03253 and SMc03254, was checked and confirmed by PCR amplification. This region contains a new gene, referred to as SMc06990, encoding a glutamine synthetase domain fused to a putative carbamoyl-phosphate synthase large chain ATP-binding protein, an enzyme that catalyzes the production of carbamoyl phosphate, which can be subsequently employed in both pyrimidine and arginine biosyntheses, 50 as well as the SMc06992 gene which is a duplication (100% identical) of the SMc03253 gene preceded by two copies of its promoter region. The promoter region of SMc03253 was previously shown to be a duplication of the whole promoter region of fixK , a gene whose expression is controlled by the key symbiotic transcription regulator FixJ.…”

System Response of Metabolic Networks in Chlamydomonas reinhardtii to Total Available Ammonium

“…Carbamoyl phosphate synthetase further recycles the ammonium released by protein breakdown and periplasmic L-amino acid oxidase. Carbamoyl phosphate is a precursor in both pyrimidine and arginine biosynthesis (86) and it directly links to other up-regulated enzymes in arginine and polyamine biosynthesis, namely N-acetyl-gamma-glutamyl-phosphate reductase, argininosuccinate synthase and argininosuccinate lyase. The latter enzymes connect citric acid cycle metabolites to amino acid metabolism via fumarate and aspartate (87).…”

Gates of Enzymes

“…A similar leakage of ammonia was caused by the G359F and G359Y mutations in carbamoyl phosphate synthetase. 103,104 Negative consequences of gate removal were also observed for the FabZ-β-hydroxyacyl-acyl carrier protein dehydratase (HpFabZ), 54 in which the Y100A mutation leaves the active site completely exposed to the bulk solvent. As a result, the acyl carrier protein binds to the HpFabZ Y100A mutant much more strongly than to the wild-type HpFabZ, decreasing the mutant enzyme’s activity by more than 50% due to the very slow dissociation of the acyl carrier protein.…”