Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.3.5.5 (CPS)
1,262 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When catalyzing the formation of MgATP and carbamate from MgADP and carbamoyl phosphate, Escherichia coli carbamoyl phosphate synthetase (CPS) binds MgADP with a large negative change in heat capacity. The magnitude of this heat capacity change is not appreciably altered by the presence of a saturating concentration of either the allosteric activator ornithine or the inhibitor UMP despite the substantial and opposing effects these ligands have on the binding affinity for MgADP. By contrast, no detectable change in heat capacity is associated with the thermodynamic coupling between MgADP and either ornithine or UMP. The sign of the apparently constant enthalpic and entropic contributions to the coupling free energy for each of these ligands is opposite that of the coupling free energy, indicating that the observed allosteric phenomenology is in net opposed by the enthalpy of the interaction and instead arises from a change in entropy of the system. IMP produces only a very small allosteric effect as indicated by a near-zero value for the MgADP-IMP coupling free energy. However, the enthalpic and entropic contributions are individually larger in absolute value for the IMP coupling than for those pertaining to the other allosteric ligands, and entropy dominates the coupling free energy above 36 degrees C, causing IMP to become an activator at high temperature. In addition, the sign of the coupling enthalpy and entropy for IMP has the same sign as the coupling enthalpy and entropy produced by ornithine, suggesting that IMP and ornithine may similarly influence the enzyme at a molecular level despite binding to different allosteric sites on the enzyme. The data are consistent with a model in which the actions of the allosteric ligands arise primarily from changes in the conformational degeneracy introduced by each ligand. With this model, one can also rationalize the failure of these allosteric ligands to substantially influence kcat.
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PMID:Allosteric effects of carbamoyl phosphate synthetase from Escherichia coli are entropy-driven. 879 75

In contrast to several other glutamine amidotransferases including asparagine synthetase, cytidine 5'-triphosphate (CTP) synthetase, carbamoyl phosphate synthetase, and phosphoribosyl pyrophosphate (PRPP) amidotransferase, guanosine monophosphate synthetase (GMPS) will not utilize hydroxylamine as an alternative nitrogen source. Instead, the enzyme is inhibited by an unknown mechanism. One untested hypothesis was that hydroxylamine serves as a substrate and intercepts a xanthosine 5'-monophosphate- (XMP-) adenylate intermediate in the enzyme active site. The nucleotide product of this substitution reaction would be N2-hydroxyguanosine 5'-monophosphate (N2-OH-GMP, 2). Here we describe the chemoenzymatic preparation of 2, via the nucleotide 2-fluoroinosine 5'-monophosphate (F-IMP, 5), and characterization of both these compounds as inhibitors of Escherichia coli GMPS. F-IMP was conceived as an electronic mimic of a reactive intermediate in the GMPS reaction but was found to bind weakly to the enzyme (IC50 > 2 mM). In contrast, N2-OH-GMP shows time-dependent inhibition and is competitive with respect to XMP (Ki = 92 nM), representing the first example of a compound that displays these kinetic properties with GMPS. The mechanism of inhibition is proposed to occur via formation of a ternary E.ATP.2 complex, followed by a rate-determining isomerization to a higher affinity complex that has a t1/2 =7.5 min. The contrast in inhibitory activity for 2-substituted purines with GMPS formulates a basis for future inhibitor design. In addition, these results complement recent structural studies of GMPS and implicate the formation of the XMP-adenylate intermediate inducing a probable conformational change that stimulates the hydrolysis of glutamine.
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PMID:N2-hydroxyguanosine 5'-monophosphate is a time-dependent inhibitor of Escherichia coli guanosine monophosphate synthetase. 989 Sep 11

A linked-function analysis of the allosteric responsiveness of carbamoyl phosphate synthetase (CPS) from E. coli was performed by following the ATP synthesis reaction at low carbamoyl phosphate concentration. All three allosteric ligands, ornithine, UMP, and IMP, act by modifying the affinity of CPS for the substrate MgADP. Individually ornithine strongly promotes, and UMP strongly antagonizes, the binding of MgADP. IMP causes only a slight inhibition at 25 degreesC. When both ornithine and UMP were varied, models which presume a mutually exclusive binding relationship between these ligands do not fit the data as well as does one which allows both ligands (and substrate) to bind simultaneously. The same result was obtained with ornithine and IMP. By contrast, the actions of UMP and IMP together must be explained with a competitive model, consistent with previous reports that UMP and IMP bind to the same site. When ornithine is bound to the enzyme, its activation dominates the effects when either UMP or IMP is also bound. The relationship of this observation to the structure of CPS is discussed.
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PMID:Allosteric dominance in carbamoyl phosphate synthetase. 993 Oct 4

Replacement by alanine of Ser-948, Thr-974 and Lys-954 of Escherichia coli carbamoyl phosphate synthetase (CPS) shows that these residues are involved in binding the allosteric inhibitor UMP and the activator IMP. The mutant CPSs are active in vivo and in vitro and exhibit normal activation by ornithine, but the modulation by both UMP and IMP is either lost or diminished. The results demonstrate that the sites for UMP and IMP overlap and that the activator ornithine binds elsewhere. Since the mutated residues were found in the crystal structure of CPS near a bound phosphate, Ser-948, Thr-974 and Lys-954 bind the phosphate moiety of UMP and IMP.
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PMID:Localization of the site for the nucleotide effectors of Escherichia coli carbamoyl phosphate synthetase using site-directed mutagenesis. 1010 Jun 29

Photoaffinity labeling with IMP was used to attach covalently this activator to its binding site of Escherichia coli carbamoyl phosphate synthetase. We now identify histidine 995 of the large enzyme subunit as the amino acid that is cross-linked with IMP. The identification was carried out by comparative peptide mapping in two chromatographic systems of peptides differentially labeled with [3H]IMP and with the labeled inhibitor [14C]UMP, followed by automated Edman degradation and radiosequence analysis. Site-directed substitution of His995 by alanine confirmed His995 to be the only amino acid in the protein forming a covalent adduct with IMP. The His995Ala mutant protein was soluble and active and exhibited normal kinetics for the activator ornithine and for the substrates in the presence of ornithine. However, the mutation selectively induced changes in the activation by IMP and the inhibition by UMP, and it abolished the photolabeling of the enzyme by IMP without affecting the photolabeling by the inhibitor UMP. Since UMP is cross-linked to Lys993 [Cervera, J., et al. (1996) Biochemistry 35, 7247-7255] only two residues upstream of the site of IMP labeling, the results provide structural evidence for earlier proposals which suggested that UMP and IMP bind in a single or overlapping site. The two residues are within the region previously proposed as the binding fold for the nucleotide effectors. In the crystal structure of the enzyme, Lys993 and His995 are exposed and line a crevice where a Pi molecule was found [Thoden, J. B., et al. (1997) Biochemistry 36, 6305-6316]. UMP and IMP appear to bind in this crevice, possibly toward the C-side of the beta-sheet in a Rossman fold. Their binding in this site is consistent with the selectivity of adduct formation of UMP with Lys993 and of IMP with His995. It is also consistent with the nonessentiality of His995 for the binding, since the interactions with other residues that line the crevice must contribute a large part of the binding energy. The lack of an effect of the mutation on the activation by ornithine is consistent with the binding of this activator in a separate site in the protein.
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PMID:Photoaffinity labeling with the activator IMP and site-directed mutagenesis of histidine 995 of carbamoyl phosphate synthetase from Escherichia coli demonstrate that the binding site for IMP overlaps with that for the inhibitor UMP. 1019 2

Carbamoyl phosphate (CP), the essential precursor of pyrimidines and arginine, is made in Escherichia coli by a single carbamoyl phosphate synthetase (CPS) consisting of 41.4 and 117.7 kDa subunits, which is feed-back inhibited by UMP and activated by IMP and ornithine. The large subunit catalyzes CP synthesis from ammonia in three steps, and binds the effectors in its 15 kDa C-terminal domain. Fifteen site-directed mutations were introduced in 13 residues of this domain to investigate the mechanism of allosteric modulation by UMP and IMP. Two mutations, K993A and V994A, decreased significantly or abolished enzyme activity, apparently by interfering with the step of carbamate synthesis, and one mutation, T974A, negatively affected ornithine activation. S948A, K954A, T974A, K993A and K993W/H995A abolished or greatly hampered IMP activation and UMP inhibition as well as the binding of both effectors, monitored using photoaffinity labeling and ultracentrifugation binding assays. V994A also decreased significantly IMP and UMP binding. L990A, V991A, H995A, G997A and G1008A had more modest effects or affected more the modulation by and the binding of one than of the other nucleotide. K993W, R1020A, R1021A and K1061A were without substantial effects. The results confirm the independence of the regulatory and catalytic centers, and also confirm functional predictions based on the X-ray structure of an IMP-CPS complex. They prove that the inhibitor UMP and the activator IMP bind in the same site, and exclude that the previously observed binding of ornithine and glutamine in this site were relevant for enzyme activation. K993 and V994 appear to be involved in the transmission of the regulatory signals triggered by UMP and IMP binding. These effectors possibly change the position of K993 and V994, and alter the intermolecular contacts mediated by the regulatory domain.
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PMID:Site-directed mutagenesis of the regulatory domain of Escherichia coli carbamoyl phosphate synthetase identifies crucial residues for allosteric regulation and for transduction of the regulatory signals. 1084 52

Ornithine is an allosteric activator of carbamoyl phosphate synthetase (CPS) from Escherichia coli. Nine amino acids in the vicinity of the binding sites for ornithine and potassium were mutated to alanine, glutamine, or lysine. The residues E783, T1042, and T1043 were found to be primarily responsible for the binding of ornithine to CPS, while E783 and E892, located within the carbamate domain of the large subunit, were necessary for the transmission of the allosteric signals to the active site. In the K loop for the binding of the monovalent cation potassium, only E761 was crucial for the exhibition of the allosteric effects of ornithine, UMP, and IMP. The mutations H781K and S792K altered significantly the allosteric properties of ornithine, UMP, and IMP, possibly by modifying the conformation of the K-loop structure. Overall, these mutations affected the allosteric properties of ornithine and IMP more than those of UMP. The mutants S792K and D1041A altered the allosteric regulation by ornithine and IMP in a similar way, suggesting common features in the activation mechanism exhibited by these two effectors.
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PMID:Dissection of the conduit for allosteric control of carbamoyl phosphate synthetase by ornithine. 1191 67

The catalytic activity of carbamoyl phosphate synthetase (CPS) from Escherichia coli is allosterically regulated by UMP, IMP, and ornithine. Thirteen amino acids within the domain that harbors the overlapping binding sites for IMP and UMP were mutated to alanine and characterized. The four residues that interact directly with the phosphate moiety of IMP in the X-ray crystal structure (K954, T974, T977, and K993) were shown to have the greatest impact on the dissociation constants for the binding of IMP and UMP and the associated allosteric effects on the kinetic constants of CPS. Of the four residues that interact with the ribose moiety of IMP (S948, N1015, T1017, and S1026), S1026 was shown to be more important for the binding of IMP than UMP. Five residues (V994, I1001, D1025, V1028, and I1029) were mutated in the region of the allosteric domain that surrounds the hypoxanthine ring of IMP. With the exception of V994A, these mutations had a modest influence on the binding and subsequent allosteric effects by UMP and IMP.
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PMID:A functional analysis of the allosteric nucleotide monophosphate binding site of carbamoyl phosphate synthetase. 1191 68

The role of residues of the ornithine activator site is probed by mutagenesis in Escherichia coli carbamoyl phosphate synthetase (CPS). Mutations E783A, E783L, E892A and E892L abolish ornithine binding, E783D and T1042V decrease 2-3 orders of magnitude and E892D decreased 10-fold apparent affinity for ornithine. None of the mutations inactivates CPS. E783 mutations hamper carbamate phosphorylation and increase K(+) and MgATP requirements, possibly by perturbing the K(+)-loop near the carbamate phosphorylation site. Mutation E892A activates the enzyme similarly to ornithine, possibly by altering the position of K891 at the opening of the tunnel that delivers the carbamate to its phosphorylation site. T1042V also influences modulation by IMP and UMP, supporting signal transmission from the nucleotide effector to the ornithine site mediated by a hydrogen bond network involving T1042. Ornithine activation of CPS may be mediated by K(+)-loop and tunnel gating changes.
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PMID:Mechanism of allosteric modulation of Escherichia coli carbamoyl phosphate synthetase probed by site-directed mutagenesis of ornithine site residues. 1194 74

Although carbamoyl-phosphate synthetases (CPSs) share sequence identity, multidomain structure, and reaction mechanism, they have varying physiological roles and allosteric effectors. Escherichia coli CPS (eCPS) provides CP for both arginine and pyrimidine nucleotide biosynthesis and is allosterically regulated by metabolites from both pathways, with inhibition by UMP and activation by IMP and ornithine. The arginine-specific CPS from Saccharomyces cerevisiae (sCPS), however, apparently responds to no allosteric effectors. We have designed and analyzed a chimeric CPS (chCPS, in which the C-terminal 136 residues of eCPS were replaced by the corresponding residues of sCPS) to define the structural basis for the allosteric nonresponsiveness of sCPS and thereby provide insight into the mechanism for allosteric selectivity and responsiveness in the other CPSs. Surprisingly, ornithine and UMP each had a significant effect on chCPS activity, and did so at concentrations that were similar to those effective for eCPS. We further found that sCPS bound both UMP and IMP and that chCPS bound IMP, although none of these interactions led to changes in enzymatic activity. These findings strongly suggest that the nonresponsive sCPS is not able to communicate occupancy of the allosteric site to the active site but does contain a latent allosteric interaction domain.
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PMID:Unmasking a functional allosteric domain in an allosterically nonresponsive carbamoyl-phosphate synthetase. 1224 18


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