Required Allosteric Effector Site for  -Acetylglutamate on Carbamoyl-Phosphate Synthetase I

McCudden, Constance R.; Powers‐Lee, Susan G.

doi:10.1074/jbc.271.30.18285

Cited by 18 publications

(13 citation statements)

References 62 publications

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“…The mutations appeared to additionally affect interaction with AGA, with mutant apparent K m values that were 7-10-fold lower than for wild type hCPS. This observation was consistent with the previous reports that the C-terminal region of CPS is involved in binding AGA (24,25). Interaction with ammonia was not significantly affected by the mutations.…”

Section: Resultssupporting

confidence: 83%

Role of Cys-1327 and Cys-1337 in Redox Sensitivity and Allosteric Monitoring in Human Carbamoyl Phosphate Synthetase

Hart

Powers‐Lee

2009

Journal of Biological Chemistry

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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.

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Section: Resultssupporting

confidence: 83%

Role of Cys-1327 and Cys-1337 in Redox Sensitivity and Allosteric Monitoring in Human Carbamoyl Phosphate Synthetase

Hart

Powers‐Lee

2009

Journal of Biological Chemistry

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“…Of these, only CPSI and II are expressed in mammals, whereas CPSIII has only been described in elasmobranchs (McCudden and Powers-Lee 1996). Moreover, in contrast to CPSI, which is mitochondrially localized, CPSII is a cytoplasmic enzyme that is mainly important in de novo polyamine synthesis, is practically absent in both enterocytes and hepatocytes, and is not recognized by the antiserum used in this study to detect CPSI.…”

mentioning

confidence: 70%

Intestinal Carbamoyl Phosphate Synthase I in Human and Rat: Expression During Development Shows Species Differences and Mosaic Expression in Duodenum of Both Species

Beers

Rings

Posthuma

et al. 1998

J Histochem Cytochem.

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SUMMARYThe clinical importance of carbamoyl phosphate synthase I (CPSI) relates to its capacity to metabolize ammonia, because CPSI deficiencies cause lethal serum ammonia levels. Although some metabolic parameters concerning liver and intestinal CPSI have been reported, the extent to which enterocytes contribute to ammonia conversion remains unclear without a detailed description of its developmental and spatial expression patterns. Therefore, we determined the patterns of enterocytic CPSI mRNA and protein expression in human and rat intestine during embryonic and postnatal development, using in situ hybridization and immunohistochemistry. CPSI protein appeared during human embryogenesis in liver at 31-35 e.d. (embryonic days) before intestine (59 e.d.), whereas in rat CPSI detection in intestine (at 16 e.d.) preceded liver (20 e.d.). During all stages of development there was a good correlation between the expression of CPSI protein and mRNA in the intestinal epithelium. Strikingly, duodenal enterocytes in both species exhibited mosaic CPSI protein expression despite uniform CPSI mRNA expression in the epithelium and the presence of functional mitochondria in all epithelial cells. Unlike rat, CPSI in human embryos was expressed in liver before intestine. Although CPSI was primarily regulated at the transcriptional level, CPSI protein appeared mosaic in the duodenum of both species, possibly due to post-transcriptional regulation. (J Histochem Cytochem 46:231-240, 1998)

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“…The allosteric effectors bind to a regulatory subdomain (Fig. 2, B3) at the carboxyl end of CPS.B in CAD (52) and other CPSases (53,54). Several studies (1) suggest that allosteric regulation of CAD governs the rate of pyrimidine biosynthesis in vivo.…”

Section: Regulation Of Pyrimidine Biosynthesismentioning

confidence: 99%

Mammalian Pyrimidine Biosynthesis: Fresh Insights into an Ancient Pathway

Evans

Guy

2004

Journal of Biological Chemistry

367

322

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Required Allosteric Effector Site for -Acetylglutamate on Carbamoyl-Phosphate Synthetase I

Cited by 18 publications

References 62 publications

Role of Cys-1327 and Cys-1337 in Redox Sensitivity and Allosteric Monitoring in Human Carbamoyl Phosphate Synthetase

Role of Cys-1327 and Cys-1337 in Redox Sensitivity and Allosteric Monitoring in Human Carbamoyl Phosphate Synthetase

Intestinal Carbamoyl Phosphate Synthase I in Human and Rat: Expression During Development Shows Species Differences and Mosaic Expression in Duodenum of Both Species

Mammalian Pyrimidine Biosynthesis: Fresh Insights into an Ancient Pathway

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