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Molecular Characterization of Carbamoyl‐Phosphate Synthetase ( CPS 1) Deficiency Using Human Recombinant CPS 1 as a Key Tool
Author(s) -
DiezFernandez Carmen,
Martínez Ana I.,
Pekkala Satu,
Barcelona Belén,
PérezArellano Isabel,
Guadalajara Ana María,
Summar Marshall,
Cervera Javier,
Rubio Vicente
Publication year - 2013
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.22349
Subject(s) - biology , carbamoyl phosphate synthetase , biochemistry , mutation , recombinant dna , microbiology and biotechnology , urea cycle , enzyme , gene , activator (genetics) , amino acid , arginine
The urea cycle disease carbamoyl‐phosphate synthetase deficiency ( CPS 1 D ) has been associated with many mutations in the CPS 1 gene [Häberle et al., 2011. Hum Mutat 32:579–589]. The disease‐causing potential of most of these mutations is unclear. To test the mutations effects, we have developed a system for recombinant expression, mutagenesis, and purification of human carbamoyl‐phosphate synthetase 1 ( CPS 1), a very large, complex, and fastidious enzyme. The kinetic and molecular properties of recombinant CPS 1 are essentially the same as for natural human CPS 1. Glycerol partially replaces the essential activator N ‐acetyl‐ l ‐glutamate ( NAG ), opening possibilities for treating CPS 1 D due to NAG site defects. The value of our expression system for elucidating the effects of mutations is demonstrated with eight clinical CPS 1 mutations. Five of these mutations decreased enzyme stability, two mutations drastically hampered catalysis, and one vastly impaired NAG activation. In contrast, the polymorphisms p.Thr344Ala and p.Gly1376Ser had no detectable effects. Site‐limited proteolysis proved the correctness of the working model for the human CPS 1 domain architecture generally used for rationalizing the mutations effects. NAG and its analogue and orphan drug N ‐carbamoyl‐ l ‐glutamate, protected human CPS 1 against proteolytic and thermal inactivation in the presence of MgATP, raising hopes of treating CPS 1 D by chemical chaperoning with N ‐carbamoyl‐ l ‐glutamate.