Functional characterization of carbamoyl phosphate synthetase I deficiency and identification of the binding site for enzyme activator

Carbamoyl phosphate synthetase I deficiency (CPSID), an autosomal, recessively inherited error of the urea cycle, causes life-threatening hyperammonemia. CPSI is a multidomain 1464-residue mitochondrial matrix protein of liver that synthesizes carbamoyl phosphate (CP) and is allosterically activated by N-acetyl-L-glutamate (NAG). In this study, we have characterized the pathogenicity and the functional impact of eleven missense mutations of CPSI, which was done by producing the recombinant CPSI in insect cells, introducing the desired mutations to the protein by site-directed mutagenesis and by studying the effects of mutations on the stability and function of CPSI. Four mutations completely abolished CP synthesis. All the remainder mutations except one, which corresponds to CPSI polymorphism, decreased CP synthesis and, in addition, two of them rendered the enzyme much less stable. The results validate the use of rat recombinant CPSI as a pathogenicity testing model for human CPSID, support the causality of the mutations, and identify the derangements caused by several of the mutated residues. We have also identified the binding site for the enzyme activator, NAG. We selected various amino acid residues for mutagenesis basing on the known binding site of allosteric effectors of homologous CPS of Escherichia coli and produced the mutant enzyme forms in insect cells. Several of the mutated residues clearly affected the activation of CPSI by NAG and seem to be implicated in allosteric signal transmission too. The crystalline structure of a fragment of the CPSI regulatory domain also enabled the screening for the cavities within the structure and docking of NAG into the structure.