Comparative steady-state fluorescence studies of cytosolic rat liver (GTP), Saccharomyces cerevisiae (ATP) and Escherichia coli (ATP) phosphoenolpyruvate carboxykinases

Encinas, M. V.; Rojas, Mireya; Goldie, Hughes; Cardemil, Emilio

doi:10.1016/0167-4838(93)90147-j

Cited by 18 publications

(11 citation statements)

References 39 publications

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“…This in turn leads to the residues from Arg-449 to Thr-455 moving toward the active site cleft, with Arg-449 and Ile-452 sandwiching the hydrophobic surface of adenine. An additional consequence of this conformational change is that Trp-443 becomes more solvent exposed when ATP-Mg 2ϩ is bound by the enzyme, a finding consistent with fluorescence studies in solution (42). An additional unique aspect of ATP binding in E. coli PCK is that it is bound in a syn rather than anti conformation, with a sugar-base torsion angle, , of 57.5° (25).…”

supporting

confidence: 59%

“…Analysis of the ternary complex of E. coli PCK is consistent with an ordered mechanism, where OAA adds prior to ATP, since the ATPinduced conformational change results in exclusion of bulk solvent from the active site and completely buries the inhibitor oxalate (25). Such a mechanism might also be expected for rat liver PCK, as this enzyme may undergo a similar change in structure upon binding NTPs (42).…”

Section: Minireview: Structure and Mechanism Of Pck 8107mentioning

confidence: 65%

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Structure and Mechanism of Phosphoenolpyruvate Carboxykinase

Matte¹,

Tari²,

Goldie

et al. 1997

Journal of Biological Chemistry

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136

108

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supporting

confidence: 59%

Section: Minireview: Structure and Mechanism Of Pck 8107mentioning

confidence: 65%

Structure and Mechanism of Phosphoenolpyruvate Carboxykinase

Matte¹,

Tari²,

Goldie

et al. 1997

Journal of Biological Chemistry

Self Cite

136

108

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“… a Metal ion concentration was 2 m m in all cases. b From [6], calculated from the Trp fluorescence quenching in the unlabeled enzyme. c This work, calculated from the Trp fluorescence quenching in the unlabeled enzyme.…”

Section: Effect Of Ligands On the P‐pyridoxyl Fluorescencementioning

confidence: 99%

“…Results obtained with AlF 3 complexes of E. coli PEP carboxykinase indicate that phosphoryl transfer occurs via a direct displacement mechanism with associative qualities [5]. In spite of the detailed knowledge of the structural characteristics of E. coli carboxykinase, very little information is available for ATP‐dependent carboxykinases with respect to thermodynamic data on ligand binding [6].…”

mentioning

confidence: 99%

Ligand interactions and protein conformational changes of phosphopyridoxyl‐labeled Escherichia coli phosphoenolpyruvate carboxykinase determined by fluorescence spectroscopy

Encinas

González-Nilo

Goldie

et al. 2002

European Journal of Biochemistry

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Escherichia coli phosphoenolpyruvate (PEP) carboxykinase catalyzes the decarboxylation of oxaloacetate and transfer of the c-phosphoryl group of ATP to yield PEP, ADP, and CO 2 . The interaction of the enzyme with the substrates originates important domain movements in the protein. In this work, the interaction of several substrates and ligands with E. coli PEP carboxykinase has been studied in the phosphopyridoxyl (P-pyridoxyl)-enzyme adduct. The derivatized enzyme retained the substrate-binding characteristics of the native protein, allowing the determination of several protein-ligand dissociation constants, as well as the role of Mg 2+ and Mn 2+ in substrate binding. The binding affinity of PEP to the enzyme-Mn 2+ complex was )8.9 kcalAEmol )1 , which is 3.2 kcalAEmol )1 more favorable than in the complex with Mg 2+ . For the substrate nucleotide-metal complexes, similar binding affinities ()6.0 to )6.2 kcalAEmol )1 ) were found for either metal ion. The fluorescence decay of the P-pyridoxyl group fitted to two lifetimes of 5.15 ns (34%) and 1.2 ns. These lifetimes were markedly altered in the derivatized enzyme-PEP-Mn complexes, and smaller changes were obtained in the presence of other substrates. Molecular models of the P-pyridoxyl-E. coli PEP carboxykinase showed different degrees of solvent-exposed surfaces for the P-pyridoxyl group in the open (substrate-free) and closed (substrate-bound) forms, which are consistent with acrylamide quenching experiments, and suggest that the fluorescence changes reflect the domain movements of the protein in solution.Keywords: Escherichia coli phosphoenolpyruvate carboxykinase; ligand binding; conformational changes; P-pyridoxyl fluorescence spectroscopy.Escherichia coli phosphoenolpyruvate carboxykinase [PEP carboxykinase; ATP:oxaloacetate carboxylase (trans-phosphorylating) EC 4.1.1.49] catalyzes the reversible decarboxylation of oxaloacetic acid (OAA) with the associated transfer of the c-phosphoryl group of ATP to yield PEP and ADP, where M 2+ is a divalent metal ion:The physiological role of this enzyme in bacteria and most other organisms is to catalyze the formation of PEP in the first committed step of gluconeogenesis [1]. The crystal structure of free-and substrate-bound E. coli PEP carboxykinase has been solved at 1.9 Å resolution [2,3]. The enzyme is a monomeric, globular protein that belongs to the a/b protein class. The overall structure has two domains, a 275 residue N-terminal domain, and a more compact 265 residue C-terminal domain, with the active site in a deep cleft between them. The recently reported crystal structure of Trypanosoma cruzi PEP-carboxykinase [4] shows remarkable similarity. Upon substrate binding, the E. coli enzyme undergoes a domain closure through a 20°rotation of the two domains towards each other, excluding bulk solvent from the active site and positioning active site residues for catalysis [3]. Results obtained with AlF 3 complexes of E. coli PEP carboxykinase indicate that phosphoryl transfer occurs via a direct displacement mec...

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“…Blue dextrin was used to determine the dead volume. Molecular masses were determined by plotting log molecular masses of standards versus K av , where (24), where ⌬I ϭ (I o Ϫ I obs ) and I obs is the observed emission following each addition of titrant with a resulting nucleotide concentration defined by [S]. The constant accounts for the fact that the saturating emission is not necessarily zero (i.e.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Soluble Domain of the ABC7 Type Transporter Atm1

Cowan

2003

Journal of Biological Chemistry

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2؉ and Co 2؉ were both found to be inhibitory at higher concentrations. The pH profile and structural comparison with HisP are consistent with a role for His and Lys in promoting the ATPase activity. Structural analysis of Atm1-C by CD spectroscopy suggested a similarity of secondary structure to that found for a prokaryotic homologue (HisP), whereas modeling of the Atm1-C tertiary structure using HisP as a template is also consistent with a similarity in tertiary structure. Atm1-C tends to form a dimer or higher aggregation state at higher concentration; however, the concentration dependence of Atm1-C on ATPase activity and the results of a Hill analysis (n app ‫؍‬ 1.1) demonstrated that there was essentially no cooperativity in ATP hydrolysis, in contrast to observations for the prokaryotic HisP transporter, which demonstrated full cooperativity for both full-length and the soluble domains. Accordingly, any cooperative response must be mediated through the transmembrane domain in the case of the eukaryotic Atm1 transporter.The ATP binding cassette (ABC) 1 transporters comprise a large family of integral membrane proteins responsible for the ATP-dependent translocation of solutes across biological membranes in both prokaryotes and eukaryotes. ABC transporters are composed of four structural domains: two nucleotide-binding domains (NBDs), which show a high degree of sequence similarity throughout the family, and two transmembrane domains that typically show six transmembrane-spanning helices (1). The structure and function of ABC-type proteins have been intensively studied (2-6). Recently, the first high resolution x-ray crystal structure of a prokaryotic NBD of histidine permease (HisP) was reported (7) and has provided insight on the structural basis for function in this class of ATP-dependent transporters. In prokaryotic systems, these transport complexes are usually constituted of individual subunits, whereas in the eukaryotic ABC transporters, they are normally formed from a single peptide chain (1), so a comparison of prokaryotic and eukaryotic systems may yield considerable insight into structure-function correlations between these discrete families.Saccharomyces cerevisiae Atm1p is an ABC transporter that is located in the mitochondrial inner membrane and has been implicated in iron-sulfur cluster assembly and maturation in the cytosol (8 -13). It is a transmembrane-spanning protein that is putatively involved in the transfer of iron-sulfur clusters from the mitochondrial matrix to the cytosol (14). In addition to the transmembrane-spanning domain, the protein also possesses a soluble nucleotide-binding domain that mediates hydrolysis of ATP to ADP. Presumably, this ATPase activity is involved in regulating the opening and closing of the channel and/or driving substrate through the channel. The human homologue of Atm1p, ABC7, displays a very high sequence homology in the soluble nucleotide binding domain. Defects in the human ABC7 gene have been shown to cause a rare type of X-linked sideroblastic anemia...

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Comparative steady-state fluorescence studies of cytosolic rat liver (GTP), Saccharomyces cerevisiae (ATP) and Escherichia coli (ATP) phosphoenolpyruvate carboxykinases

Cited by 18 publications

References 39 publications

Structure and Mechanism of Phosphoenolpyruvate Carboxykinase

Structure and Mechanism of Phosphoenolpyruvate Carboxykinase

Ligand interactions and protein conformational changes of phosphopyridoxyl‐labeled Escherichia coli phosphoenolpyruvate carboxykinase determined by fluorescence spectroscopy

Characterization of the Soluble Domain of the ABC7 Type Transporter Atm1

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