A Molecular Wedge for Triggering the Amidotransferase Activity of Carbamoyl Phosphate Synthetase

Mareya, Shadreck M.; Raushel, Frank M.

doi:10.1021/bi00176a026

Cited by 43 publications

(62 citation statements)

References 19 publications

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“…The mutant plasmids were transformed in the E. coli RC50 cell line for the expression of the modified proteins. The wild-type and mutant proteins were purified as described by Mareya et al (24).…”

Section: Mutagenesis Expression and Purification Of The Mutant Protementioning

confidence: 99%

“…The synthesis of carbamoyl phosphate was determined by measuring the rate of citrulline formation in a coupled assay containing ornithine 7 transcarbamoylase and ornithine (25). The rate of ATP hydrolysis was determined by assaying the formation of MgADP using a pyruvate kinase/lactate dehydrogenase coupling system (24).…”

Section: Mutagenesis Expression and Purification Of The Mutant Protementioning

confidence: 99%

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Restricted Passage of Reaction Intermediates through the Ammonia Tunnel of Carbamoyl Phosphate Synthetase

Huang

Raushel

2000

Journal of Biological Chemistry

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SUMMARYThe x-ray crystal structure of the heterodimeric carbamoyl phosphate synthetase from Escherichia coli has identified an intermolecular tunnel that connects the glutamine binding site within the small amidotransferase subunit to the two phosphorylation sites within the large synthetase subunit. The tunneling of the ammonia intermediate through the interior of the protein has been proposed as a mechanism for the delivery of the ammonia from the small subunit to the large subunit. A series of mutants created within the ammonia tunnel were prepared by the placement of a constriction via site-directed mutagenesis. The degree of constriction within the ammonia tunnel of these enzymes was found to correlate to the extent of the uncoupling of the partial reactions, the diminution of carbamoyl phosphate formation, and the percentage of the internally-derived ammonia that is channeled through the ammonia tunnel.NMR spectroscopy and a radiolabeled probe were used to detect and identify the enzymatic synthesis of N-amino carbamoyl phosphate and N-hydroxy carbamoyl phosphate from hydroxylamine and hydrazine. The kinetic results indicate that hydroxylamine, derived from the hydrolysis of γ-glutamyl hydroxamate, is channeled through the ammonia tunnel to the large subunit. Discrimination between the passage of ammonia and hydroxylamine was observed among some of these tunnel-impaired enzymes. The overall results provide biochemical evidence for the tunneling of ammonia within the native carbamoyl phosphate synthetase.

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Section: Mutagenesis Expression and Purification Of The Mutant Protementioning

confidence: 99%

Section: Mutagenesis Expression and Purification Of The Mutant Protementioning

confidence: 99%

Restricted Passage of Reaction Intermediates through the Ammonia Tunnel of Carbamoyl Phosphate Synthetase

Huang

Raushel

2000

Journal of Biological Chemistry

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“…CPS was purified via a modification of that presented by Mareya and Raushel (29). Cells were suspended in a 100 mM phosphate buffer, pH 7.5, containing 1 mM EDTA.…”

Section: Cps Purificationmentioning

confidence: 99%

Resolving the Fluorescence Response of Escherichia coli Carbamoyl Phosphate Synthetase: Mapping Intra- and Intersubunit Conformational Changes

et al. 2006

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Carbamoyl phosphate synthetase (CPS) from E. coli is potentially overlaid with a network of allosterism, interconnecting active sites, effector binding sites, and aggregate interfaces to control its mechanisms of catalytic synchronization, regulation, and oligomerization, respectively. To characterize these conformational changes, a tryptophan-free variant of CPS was genetically engineered by substituting six native tryptophans with tyrosines. Each tryptophan was then reinserted, singly, as a specific fluorescence probe of its corresponding microenvironment. The amino acid substitutions themselves result in little apparent disruption of the protein; variants maintain catalytic and allosteric functionality, and the fluorescence properties of each tryptophan, while unique, are additive to wild-type CPS. Whereas the collective, intrinsic fluorescence response of E. coli CPS is largely insensitive to ligand binding, changes of the individual probes in intensity, lifetime, anisotropy, and accessibility to acrylamide quenching highlight the dynamic interplay between several protein domains, as well as between subunits. W213 within the carboxy phosphate domain, for example, exhibits an almost 40% increase in intensity upon saturation with ATP; W437 of the oligomerization domain, in contrast, is essentially silent in its fluorescence to the binding of ligands. Nucleotide and bicarbonate association within the large subunit induce fluorescence changes in both W170 and W175 of the small subunit, indicative of the type of long-range interactions purportedly synchronizing the carboxy phosphate and amidotransferase domains of the enzyme to initiate catalysis. ATP and ADP engender different fluorescence responses in most tryptophans, perhaps reflecting coordinating, conformational changes accompanying the cycling of reactants and products during catalysis. † This work was supported by NIH grant P20 RR016478 (to JLJ) from the INBRE Program of the National Center for Research Resources at Southwestern Oklahoma State University, and by NIH grant GM33216 and Welch Foundation grant A1543 (to GDR) at Texas A&M University. Molecular graphics images were produced using the UCSF package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, which is supported by NIH grant P41 RR-01081. *Corresponding authors. Phone: (580) Fax: (580) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCarbamoyl phosphate synthetase (CPS) 1 from E. coli coordinates five substrates (2 ATP, bicarbonate, glutamine, and water) through a series reactions involving at least three unstable intermediates during the synthesis of its ultimate product, carbamoyl phosphate (1,2). Moreover, crystal structure coordinates have revealed that the reaction mechanism involves three distinct active sites separated by almost 100Å, but connected by a series of intra-and inter-molecular tunnels that presumably shuttle unstable intermediates from site to site (3-5).In the minimal mecha...

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“…The majority of the mutant proteins were purified by the standard protocol as described previously by Mareya and Raushel (27). In some cases DNase I was used in place of protamine sulfate to precipitate DNA, and additional inhibitors were added to inactivate cellular proteases.…”

Section: Chemicals and Enzymesmentioning

confidence: 99%

“…Determination of Enzymatic Activity-The enzymatic activity for the forward and reverse reactions were conducted as described previously (27). The formation of carbamoyl phosphate was determined by measuring the amount of citrulline formed in a coupled assay with ornithine transcarbamoylase and ornithine (28).…”

Section: Chemicals and Enzymesmentioning

confidence: 99%

The Differentially Conserved Residues of Carbamoyl-Phosphate Synthetase

Javid-Majd¹,

Mullins

Raushel

et al. 2000

Journal of Biological Chemistry

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Carbamoyl-phosphate synthetase (CPS) from Escherichia coli is a heterodimeric protein. The larger of the two subunits (M r ϳ118,000) contains a pair of homologous domains of approximately 400 residues each that are ϳ40% identical in amino acid sequence. The carboxy phosphate (residues 1-400) and carbamoyl phosphate domains (residues 553-933) also contain ϳ79 differentially conserved residues. These are residues that are conserved throughout the bacterial evolution of CPS in one of these homologous domains but not the other. The role of these differentially conserved residues in the structural and catalytic properties of CPS was addressed by swapping segments of these residues from one domain to the other. Nine of these chimeric mutant enzymes were constructed, expressed, purified, and characterized. A majority of the mutants were unable to synthesize any carbamoyl phosphate and the rest were severely crippled. True tandem repeat chimeric proteins were constructed by the complete substitution of one homologous domain sequence for the other. Neither of the two possible chimeric proteins was structurally stable. These results have been interpreted to demonstrate that the two homologous domains in the large subunit of CPS are functionally and structurally nonequivalent. This nonequivalence is a direct result of the specific functions each of these domains must perform during the overall synthesis of carbamoyl phosphate in the wild type enzyme and the specific structural alterations imposed by the differentially conserved residues. Carbamoyl-phosphate synthetase (CPS)1 is a complicated protein. This enzyme catalyzes the formation of carbamoyl phosphate via a reaction mechanism that requires two molecules of ATP, bicarbonate, and either NH 4 ϩ or glutamine as a source of ammonia. The full reaction is presented in equation 1.

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A Molecular Wedge for Triggering the Amidotransferase Activity of Carbamoyl Phosphate Synthetase

Cited by 43 publications

References 19 publications

Restricted Passage of Reaction Intermediates through the Ammonia Tunnel of Carbamoyl Phosphate Synthetase

Restricted Passage of Reaction Intermediates through the Ammonia Tunnel of Carbamoyl Phosphate Synthetase

Resolving the Fluorescence Response of Escherichia coli Carbamoyl Phosphate Synthetase: Mapping Intra- and Intersubunit Conformational Changes

The Differentially Conserved Residues of Carbamoyl-Phosphate Synthetase

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