Abstract
Human carbamoyl phosphate synthetase (hCPS) has evolved critical features that allow it to remove excess and potentially neurotoxic ammonia via the urea cycle, including use of only free ammonia as a nitrogen donor, a K(m) for ammonia 100-fold lower than for CPSs that also use glutamine as a nitrogen donor, and required allosteric activation by N-acetylglutamate (AGA), a sensor of excess amino acids. The recent availability of a Schizosaccharomyces pombe expression system for hCPS allowed us to utilize protein engineering approaches to elucidate the distinctive hCPS properties. Although the site of AGA interaction is not defined, it is known that the binding of AGA to CPS leads to a conformational change in which a pair of cysteine side chains become proximate and can then be selectively induced to undergo disulfide bonding. We analyzed the response of hCPS cysteine mutants to thiol-specific reagents and identified Cys-1327 and Cys-1337 as the AGA-responsive proximate cysteines. Possibly two of the features unique to urea-specific CPSs, relative to other CPSs (the conserved Cys-1327/Cys-1337 pair and the occurrence at very high concentrations in the liver mitochondrial matrix) co-evolved to provide buffering against reactive oxygen species. Reciprocal mutation analysis of Escherichia coli CPS (eCPS), creating P909C and G919C and establishing the ability of these engineered cysteine residues to share a disulfide bond, indicated an eCPS conformational change at least partly similar to the hCPS conformational change induced by AGA. These findings strongly suggested an alternative eCPS conformation relative to the single crystal conformation thus far identified.
Highlights
Carbamoyl phosphate synthetase (CPS) from all species with varied metabolic roles share strong sequence identity, and all appear to have the same overall domain organization [6] (Fig. 1) observed in the crystal structure of E. coli CPS, the only solved CPS structure [7]
Effects of C1327A and C1337A Substitutions on the Function of Human carbamoyl phosphate synthetase (hCPS)—Previous studies have established that a pair of cysteine side chains become proximate upon AGA binding to urea-specific CPS and that the cysteine residues can be selectively induced to undergo disulfide bonding [10, 11]. This single pair of proximate sulfhydryl groups is uniquely modified when the AGA1⁄7CPS complex is exposed to a variety of disulfideinducing reagents, and reversible activity loss accompanies disulfide formation. The specificity of these reactions must depend on the relative unreactivity or inaccessibility of the other 18 hCPS cysteinyl residues
The effect of the mutations themselves on the function of hCPS was determined by analyzing the overall and partial reactions catalyzed by the enzyme
Summary
Recombinant DNA Methods—Bacterial transformations and recombinant DNA techniques were carried out as described in Sambrook et al [13]. CPS Expression and Purification—The protocol for expression and purification of recombinant wild type eCPS and for its site-directed mutants was as previously described [14]. The reaction mixtures contained 50 mM HEPES, 100 mM KCl, 10 mM ATP, 20 mM MgCl2, 20 mM NaHCO3, 5 mM ornithine, 0.2 units ornithine transcarbamoylase, and either 300 mM NH4Cl or 10 mM glutamine and were initiated by the addition of CPS (final volume 100 l, pH 7.6). Rates of inactivation were determined by adding 10-l aliquots of the reaction mixture to the CP synthesis activity assay mixture (final reaction volume of 100 l, pH 7.6) at the indicated times and incubating at 30 °C for 20 min. Visualization of eCPS Crystal Structure—Figures which include representations of the eCPS crystal structure were created using the University of California, San Francisco Chimera [20] and Friend [21] programs
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