Precision medicine in rare disease: Mechanisms of disparate effects of N-carbamyl-l-glutamate on mutant CPS1 enzymes.

This study documents the disparate therapeutic effect of N-carbamyl-l-glutamate (NCG) in the activation of two different disease-causing mutants of carbamyl phosphate synthetase 1 (CPS1). We investigated the effects of NCG on purified recombinant wild-type (WT) mouse CPS1 and its human corresponding E1034G (increased ureagenesis on NCG) and M792I (decreased ureagenesis on NCG) mutants. NCG activates WT CPS1 sub-optimally compared to NAG. Similar to NAG, NCG, in combination with MgATP, stabilizes the enzyme, but competes with NAG binding to the enzyme. NCG supplementation activates available E1034G mutant CPS1 molecules not bound to NAG enhancing ureagenesis. Conversely, NCG competes with NAG binding to the scarce M792I mutant enzyme further decreasing residual ureagenesis. These results correlate with the respective patient's response to NCG. Particular caution should be taken in the administration of NCG to patients with hyperammonemia before their molecular bases of their urea cycle disorders is known.

Down-regulation of hepatic urea synthesis by oxypurines: xanthine and uric acid inhibit N-acetylglutamate synthase.

We previously reported that isobutylmethylxanthine (IBMX), a derivative of oxypurine, inhibits citrulline synthesis by an as yet unknown mechanism. Here, we demonstrate that IBMX and other oxypurines containing a 2,6-dione group interfere with the binding of glutamate to the active site of N-acetylglutamate synthetase (NAGS), thereby decreasing synthesis of N-acetylglutamate, the obligatory activator of carbamoyl phosphate synthase-1 (CPS1). The result is reduction of citrulline and urea synthesis. Experiments were performed with (15)N-labeled substrates, purified hepatic CPS1, and recombinant mouse NAGS as well as isolated mitochondria. We also used isolated hepatocytes to examine the action of various oxypurines on ureagenesis and to assess the ameliorating affect of N-carbamylglutamate and/or l-arginine on NAGS inhibition. Among various oxypurines tested, only IBMX, xanthine, or uric acid significantly increased the apparent K(m) for glutamate and decreased velocity of NAGS, with little effect on CPS1. The inhibition of NAGS is time- and dose-dependent and leads to decreased formation of the CPS1-N-acetylglutamate complex and consequent inhibition of citrulline and urea synthesis. However, such inhibition was reversed by supplementation with N-carbamylglutamate. The data demonstrate that xanthine and uric acid, both physiologically occurring oxypurines, inhibit the hepatic synthesis of N-acetylglutamate. An important and novel concept emerging from this study is that xanthine and/or uric acid may have a role in the regulation of ureagenesis and, thus, nitrogen homeostasis in normal and disease states.

Identification, cloning and expression of the mouse N-acetylglutamate synthase gene.

In ureotelic animals, N-acetylglutamate (NAG) is an essential allosteric activator of carbamylphosphate synthetase I (CPSI), the first enzyme in the urea cycle. NAG synthase (NAGS; EC 2.3.1.1) catalyses the formation of NAG from glutamate and acetyl-CoA in liver and intestinal mitochondria. This enzyme is supposed to regulate ureagenesis by producing variable amounts of NAG, thus modulating CPSI activity. Moreover, inherited deficiencies in NAGS have been associated with hyperammonaemia, probably due to the loss of CPSI activity. Although the existence of the NAGS protein in mammals has been known for decades, the gene has remained elusive. We identified the mouse (Mus musculus) and human NAGS genes using their similarity to the respective Neurospora crassa gene. NAGS was cloned from a mouse liver cDNA library and was found to encode a 2.3 kb message, highly expressed in liver and small intestine with lower expression levels in kidney, spleen and testis. The deduced amino acid sequence contains a putative mitochondrial targeting signal at the N-terminus. The cDNA sequence complements an argA (NAGS)-deficient Escherichia coli strain, reversing its arginine auxotrophy. His-tagged versions of the pre-protein and two putative mature proteins were each overexpressed in E. coli, and purified to apparent homogeneity by using a nickel-affinity column. The pre-protein and the two putative mature proteins catalysed the NAGS reaction but one of the putative mature enzymes had significantly higher activity than the pre-protein. The addition of l-arginine increased the catalytic activity of the purified recombinant NAGS enzymes by approx. 2-6-fold.