Human carbamoyl-phosphate synthetase: Insight into N-acetylglutamate interaction and the functional effects of a common single nucleotide polymorphism
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Human carbamoyl-phosphate synthetase (hCPS) has evolved three features that allow it to remove excess, potentially neurotoxic ammonia via the urea cycle: inability to use glutamine as an alternative nitrogen donor; a K m for ammonia 100-fold lower than for CPSs that also use glutamine; and required allosteric activation by N-acetylglutamate (AGA), a sensor of excess amino acids. To determine the structural features of hCPS that allow its unique functioning, we have developed the first recombinant expression system for hCPS, utilizing Schizosaccharomyces pombe. Of several common single-nucleotide polymorphisms identified in the gene encoding hCPS, only the one resulting in substitution of threonine at position 1406 with asparagine has been linked to phenotypic effects. We have expressed and characterized both variants of hCPS. The asparagine polymorph, hCPS_N, consistently displayed inferior catalytic properties, but the K m and k cat values for overall and partial reactions varied only by a factor of 1.7 or less. We have designed and characterized an hCPS construction from which the N-terminal domain A is deleted. hCPS_ΔA was competent to bind AGA, demonstrating that domain A does not contain the AGA binding site. Thus, the site at the C/D boundary previously identified by AGA analogue labelling appears to be the functionally significant initial binding site for AGA. However, hCPS_ΔA was not able to fully assume the catalytically competent conformation, with specific activity of CP formation decreased 700-fold.
KeywordsCarbamoyl Phosphate Carbamoyl Phosphate Synthetase Partial cDNA Clone Recombinant Expression System Carbamyl Phosphate Synthetase
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