Limited proteolysis reveals low‐affinity binding of <i>N</i>‐acetyl‐L‐glutamate to rat‐liver carbamoyl‐phosphate synthetase (ammonia)

Guadalajara, Ana María; Grisolı́a, Santiago; Rubio, Vicente

doi:10.1111/j.1432-1033.1987.tb11207.x

Cited by 13 publications

(8 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Limited proteolysis has been used to delineate the structural organization of several amidotransferases including asparagine synthase [43], carbamoyl phosphate synthase [44–50], anthranilate synthase [51], and glucosamine‐6‐phosphate synthase [52]. This methodology has been particularly useful for identifying both ligand‐binding sites and, in the case with glucosamine‐6‐phosphate synthase, an exposed ‘hinge’ region that, when cleaved by α‐chymotrypsin, led to separation of the enzyme into its GAT and synthase domains.…”

Section: Discussionmentioning

confidence: 99%

Limited proteolysis of Escherichia coli cytidine 5′‐triphosphate synthase. Identification of residues required for CTP formation and GTP‐dependent activation of glutamine hydrolysis

Simard

Hewitt

Lunn

et al. 2003

European Journal of Biochemistry

View full text Add to dashboard Cite

Cytidine 5¢-triphosphate synthase catalyses the ATPdependent formation of CTP from UTP using either ammonia or L-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis. Lys187 is located at the CTP/UTP-binding site within the synthase domain, and cleavage at this site destroyed all synthase activity. Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP). The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP. K187A was able to form tetramers in the presence of UTP and ATP. Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer (GAT) domain. Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage. The R429A and R429A/ K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH 3 -dependent CTP formation. For these mutants, the values of k cat /K m and k cat for glutamine-dependent CTP formation were reduced % 20-fold and % 10-fold, respectively, relative to wild-type enzyme; however, the value of K m for glutamine was not significantly altered. Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold. This suggests that Arg429 plays a role in both GTP-dependent activation and GTP binding.Keywords: activation; amidotransferase; CTP synthase; glutaminase; proteolysis; site-directed mutagenesis.CTP synthase [CTPS; EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)] catalyses the ATP-dependent formation of CTP from UTP using either L-glutamine or NH 3 as the nitrogen source (Scheme 1) [1,2]. This glutamine amidotransferase is a single polypeptide chain containing 545 amino acids and consisting of two domains. The C-terminal glutamine amide transfer (GAT) domain catalyses the hydrolysis of glutamine, and the nascent NH 3 derived from glutamine hydrolysis is transferred to the N-terminal synthase domain where the amination of UTP is catalysed [3,4]. CTPS belongs to the Triad family of glutamine amidotransferases [5,6] which utilizes a Cys-His-Glu triad to catalyse glutamine hydrolysis and also includes anthranilate synthase, carbamoyl phosphate synthase, formylglycinamidine synthase, GMP synthase, imidazole glycerol phosphate synthase, and aminodeoxychorismate synthase.CTPS catalyses the final step in the de novo synthesis of cytosine nucleotides. Because CTP has a central role in the biosynthesis of nucleic acids [7] and membrane phospholipids [8], CTPS is a recog...

show abstract

Section: Discussionmentioning

confidence: 99%

Limited proteolysis of Escherichia coli cytidine 5′‐triphosphate synthase. Identification of residues required for CTP formation and GTP‐dependent activation of glutamine hydrolysis

Simard

Hewitt

Lunn

et al. 2003

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…We have shown that this site may adopt high-and low-affinity conformations [2,6,71. The former is adopted in the central complex [2], and thus predominates under the conditions of ~131.…”

Section: Discussionmentioning

confidence: 99%

A structure‐reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I

Britton

García‐Espanña

Goya

et al. 1990

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

The requirements for binding at the N-acetyl-L-glutamate binding site of carbamoyl phosphate synthetase I were studied by the displacement of the activator from the central enzyme complex by analogs. Two carboxyls are essential and the acetamido group, if linked to the a-carbon, enhances binding 5000-fold. The subsite for the 6-carboxyl is mobile with respect to that for the a-carboxyl. Mixtures of complementary fragment of acetylglutamate do not bind, indicating a strong 'chelate' effect. Substituents revealed the existence of steric constraints around the 6-carboxyl, the a and y-carbons, and the whole of the acetamido group. However, phenyl substituents at the P-carbon did not hamper binding, indicating that substituents at the P-carbon face the solution. This is consistent with binding of acetylglutamate as the minimum-energy conformer. All analogs binding with high affinity are activators. Some analogs that bind poorly are competitive inhibitors. They appear to bind preferentially to a low-affinity conformation adopted by the site when the products dissociate and the substrates bind. The acetamido group plays no role in the binding of the inhibitors but it is crucial for the binding of the activators, and the high-and low-affinity conformations of the site differ markedly in structural selectivity. from ATPA which is the ATP molecule yielding Pi and ATPB is the ATP molecule yielding carbamoyl phosphate) is considerably greater than for the free enzyme [2, 6, 71. The stereospecificity indicates that at least three of the groups attached to the asymmetric a-carbon of acetylglutamate are involved in the binding. Information about the specific role of these groups should be of value in designing agonists and antagonists of acetylglutamate and in mapping the binding site, which is located in the 20-kDa COOH-terminal domain of the enzyme [8].In the present work, binding studies with analogs of acetylglutamate, in which the groups attached to the asymmetric carbon have been systematically modified, have provided acetylglutamate

show abstract

“…SDS-PAGE analysis indicated Ն 95% purity for all constructions. Because protease treatments necessary to remove fusion partners also result in proteolysis of fCPS, presumably at the links between domains (13)(14)(15)(16), in the present studies we retained the tag in all fCPS constructions.…”

Section: Methodsmentioning

confidence: 99%

Gain of Glutaminase Function in Mutants of the Ammonia-specific Frog Carbamoyl Phosphate Synthetase

Saeed-Kothe

Powers‐Lee

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Depending on their physiological role, carbamoyl phosphate synthetases (CPSs) use either glutamine or free ammonia as the nitrogen donor for carbamoyl phosphate synthesis. Sequence analysis of known CPSs indicates that, regardless of whether they are ammonia-or glutamine-specific, all CPSs contain the structural equivalent of a triad-type glutamine amidotransferase (GAT) domain. In ammonia-specific CPSs, such as those of rat or human, the catalytic inactivity of the GAT domain can be rationalized by the substitution of the Triad cysteine residue by serine (1). The ammonia-specific CPS of Rana catesbeiana (fCPS) presents an interesting anomaly in that, despite its retention of the entire catalytic triad (2) and almost all other residues conserved in Triad GATs, it is unable to utilize glutamine as a nitrogen-donating substrate (3). Based on our earlier work with the glutamine-utilizing E. coli CPS (eCPS), we have targeted residues Lys 258 and Glu 261 in the fCPS GAT domain as critical for preventing GAT function. Previously we have shown that substitution of the corresponding residues in eCPS by their fCPS counterparts (Leu 3 Lys and Gln 3 Glu) resulted in complete loss of GAT function in eCPS (3). To examine the role of these residues in the fCPS GAT component, we have cloned the full-length fCPS gene from R. catesbeiana liver. Here we report the first heterologous expression of an ammonia-specific CPS and show that a single mutation of the frog enzyme, K258L, yields a gain of glutaminase function.

show abstract

Limited proteolysis reveals low‐affinity binding of N‐acetyl‐L‐glutamate to rat‐liver carbamoyl‐phosphate synthetase (ammonia)

Cited by 13 publications

References 24 publications

Limited proteolysis of Escherichia coli cytidine 5′‐triphosphate synthase. Identification of residues required for CTP formation and GTP‐dependent activation of glutamine hydrolysis

Limited proteolysis of Escherichia coli cytidine 5′‐triphosphate synthase. Identification of residues required for CTP formation and GTP‐dependent activation of glutamine hydrolysis

A structure‐reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I

Gain of Glutaminase Function in Mutants of the Ammonia-specific Frog Carbamoyl Phosphate Synthetase

Contact Info

Product

Resources

About