Critical Roles for Arginine 1061/1060 and Tyrosine 1057 inSaccharomyces cerevisiaeArginine-Specific Carbamoyl-Phosphate Synthetase

Lim, Angela L; Powers‐Lee, Susan G.

doi:10.1006/abbi.1997.9887

Cited by 5 publications

(3 citation statements)

References 61 publications

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“…In any case, mutations affecting this invariant arginine (R1453 in human) should be inactivating only in CPS1, because other CPSs are active in the absence of any effectors. Indeed, mutation to alanine of the corresponding arginine of EcCPS and of yeast CPS (R1030A and R1060A mutations, respectively) fails to inactivate these CPSs [Czerwinski et al, 1995;Lim and Powers-Lee, 1997].…”

Section: Pg1376s Is a Polymorphism With No Functional Consequencesmentioning

confidence: 99%

Understanding carbamoyl-phosphate synthetase I (CPS1) deficiency by using expression studies and structure-based analysis

et al. 2010

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Carbamoyl‐phosphate synthetase I (CPS1) deficiency (CPS1D), a recessively inherited urea cycle error due to CPS1 gene mutations, causes life‐threatening hyperammonemia. The disease‐causing potential of missense mutations in CPS1 deficiency can be ascertained with the recombinant CPS1 expression and purification system reported here, which uses baculovirus and insect cells. We study with this system the effects of nine clinical mutations and one polymorphism on CPS1 solubility, stability, activity, and kinetic parameters for NAG. Five of the mutations (p.T471N, p.Q678P, p.P774L, p.R1453Q, and p.R1453W) are first reported here, in three severe CPS1D patients. p.P774L, p.R1453Q, and p.R1453W inactivate CPS1, p.T471N and p.Y1491H greatly decrease the apparent affinity for NAG, p.Q678P hampers correct enzyme folding, and p.S123F, p.H337R, and p.P1411L modestly decrease activity. p.G1376S is confirmed a trivial polymorphism. The effects of the C‐terminal domain mutations are rationalized in the light of this domain crystal structure, including the NAG site structure [Pekkala et al. Biochem J 424:211–220]. The agreement of clinical observations and in vitro findings, and the possibility to identify CPS1D patients who might benefit from specific treatment with NAG analogues because they exhibit reduced affinity for NAG highlight the value of this novel CPS1 expression/purification system. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.

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Section: Pg1376s Is a Polymorphism With No Functional Consequencesmentioning

confidence: 99%

Understanding carbamoyl-phosphate synthetase I (CPS1) deficiency by using expression studies and structure-based analysis

et al. 2010

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“…Standard yeast genetic and molecular biological procedures were performed as described (37). Site-directed mutagenesis was performed by the recombinant PCR method (38) or the unique site-elimination method (39) as described for yeast arginine-specific CPS (33,40). The presence of desired mutations and the absence of any unexpected mutations in each mutagenesis cassette was confirmed by sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…To determine whether mutant constructions could produce CP, their ability to functionally complement CPS-disrupted yeast strain LPL26 was assessed (41). Generation time determination, crude extract preparation, enzymatic activity assay, protein determination, SDS͞PAGE, and Western blot analysis were as previously utilized for CPS mutational analysis (33,40 …”

Section: Methodsmentioning

confidence: 99%

Novel mechanism for carbamoyl-phosphate synthetase: A nucleotide switch for functionally equivalent domains

Kothe

Eroglu

Mazza

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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We have carried out a site-directed mutagenic analysis that is consistent with ATP binding to a palmate motif rather than to a Walker A͞B motif in domains B and C. To accommodate our present findings, as well as the other recent findings of structural and functional equivalence, we are proposing a novel mechanism for CPS. In this mechanism utilization of ATP bound to domain C is coupled to carbamoylphosphate synthesis at domain B via a nucleotide switch, with the energy of ATP hydrolysis at domain C allowing domain B to cycle between two alternative conformations.Carbamoyl-phosphate (CP) is a high-energy biological compound that plays a key role in the introduction of both ammonia and single carbon units into the metabolic pool. CP may serve either as the precursor for synthesis of pyrimidine nucleotides or, in an alternative pathway, as the precursor for arginine. In addition to serving as a required component of almost all proteins, arginine is used in the liver for ammonia detoxification via the urea cycle and, under appropriate physiological conditions, it is also used in a variety of tissues for nitric oxide formation. Depending on the physiological role, CP synthetases (CPSs) vary in mode of regulation and subunit composition (1). However, all of the CPSs thus far sequenced display marked primary sequence identity (2, 3), and all catalyze the formation of CP, P i , and two ADP from HCO 3 Ϫ , NH 3 , and two ATP. Sequence analysis demonstrated regions of internal duplication within CPS and suggested, on the basis of similarities to the ATP-binding sequence motifs of Walker A͞B proteins (i.e., proteins involving a ␤␣␤ nucleotide binding fold; ref. 4), that each region of internal duplication contains one ATP site (5, 6). Subsequent studies (7-10) demonstrated that the synthetase duplications correspond to independently folded domains (B and C) and that binding of one ATP molecule is localized to each of the two synthetase domains (6,(11)(12)(13). A great deal of experimental evidence (14-23) has established that the role of one ATP is to form the enzyme-bound intermediate carboxy-phosphate (CxP) and that domain B binds this ATP (ATP B ) whereas domain C binds the other ATP molecule (ATP C ).When we carried out the presently described studies, further definition of the utilization of ATP B and ATP C by CPS could not be based on a solved three-dimensional structure because there was none available for any CPS at that time. However, we found that domains B and C of CPS could each be fit to the structural coordinates for the biotin carboxylase (BN) component of Escherichia coli acetyl Co-A carboxylase (24). This BN homology modeling seemed appropriate for the following reasons. First, there is significant sequence identity shared by portions of BN (as well as other biotin-dependent enzymes) and each of the CPS domains B and C (2). Also, both CPS and BN couple cleavage of ATP to ADP with the activation of HCO 3 Ϫ to yield CxP, and both catalyze reaction of the activated carboxyl group with an amino group (NH 3 for...

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Identification of critical amino acid residues of Saccharomyces cerevisiae carbamoyl-phosphate synthetase: definition of the ATP site involved in carboxy-phosphate formation

Zheng¹,

Lim²,

Powers‐Lee³

1997

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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Critical Roles for Arginine 1061/1060 and Tyrosine 1057 inSaccharomyces cerevisiaeArginine-Specific Carbamoyl-Phosphate Synthetase

Cited by 5 publications

References 61 publications

Understanding carbamoyl-phosphate synthetase I (CPS1) deficiency by using expression studies and structure-based analysis

Understanding carbamoyl-phosphate synthetase I (CPS1) deficiency by using expression studies and structure-based analysis

Novel mechanism for carbamoyl-phosphate synthetase: A nucleotide switch for functionally equivalent domains

Identification of critical amino acid residues of Saccharomyces cerevisiae carbamoyl-phosphate synthetase: definition of the ATP site involved in carboxy-phosphate formation

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