Urea-induced unfolding studies of free- and ligand-bound tetrameric ATP-dependent Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: Influence of quaternary structure on protein conformational stability

M. Victoria Encinas, Fernando D. González-Nilo, José M. Andreu, Carlos Alfonso, Emilio Cardemil

Resultado de la investigación: Article

5 Citas (Scopus)

Resumen

ATP-dependent phosphoenolpyruvate (PEP) carboxykinases are found in plants and microorganisms, and catalyse the reversible formation of PEP, ADP, and CO2 from oxaloacetate plus ATP. These enzymes vary in quaternary structure although there is significant sequence identity among the proteins isolated from different sources. To help understand the influence of quaternary structure in protein stability, the urea-induced unfolding of free- and substrate-bound tetrameric Saccharomyces cerevisiae PEP carboxykinase is described and compared with the unfolding characteristics of the monomeric Escherichia coli enzyme [Eur. J. Biochem. 255 (1998) 439]. The urea-induced denaturation of S. cerevisiae PEP carboxykinase was studied by monitoring the enzyme activity, intrinsic protein fluorescence, circular dichroism (CD) spectra, and 1-anilino-8-naphthalenesulfonate (ANS) binding. The unfolding profiles were multi-steps, and formation of hydrophobic structures were detected. The data indicate that unfolding and dissociation of the enzyme tetramer are simultaneous events. Ligand binding, most notably PEP in the presence of MnCl2, conferred a marked protection against urea-induced denaturation. A similar protection effect was found when N-iodoacetyl-N′-(5-sulfo-1-napthyl)ethylene diamine (1,5-I-AEDANS) was covalently bound at Cys365, within the active site region. Refolding experiments indicated that total recovery of tertiary structure was only obtained from samples previously unfolded to less than 30%. In the presence of substrates, complete refolding was achieved from samples originally denatured up to 50%. The unfolding behaviour of S. cerevisiae PEP carboxykinase was found to be similar to that of E. coli PEP carboxykinase, however all steps take place at lower urea concentrations. These findings show that, at least for monomeric and tetrameric ATP-dependent PEP carboxykinases, quaternary structure does not contribute to protein conformational stability.

Idioma originalEnglish
Páginas (desde-hasta)645-656
Número de páginas12
PublicaciónInternational Journal of Biochemistry and Cell Biology
Volumen34
N.º6
DOI
EstadoPublished - 13 abr 2002

Huella dactilar

Phosphoenolpyruvate
Protein Stability
Yeast
Saccharomyces cerevisiae
Urea
Adenosine Triphosphate
Ligands
Denaturation
Escherichia coli
Proteins
Enzymes
Oxaloacetic Acid
Diamines
Enzyme activity
Substrates
Microorganisms
Phosphoenolpyruvate Carboxykinase (ATP)
Adenosine Diphosphate
Fluorescence
Recovery

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Citar esto

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title = "Urea-induced unfolding studies of free- and ligand-bound tetrameric ATP-dependent Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: Influence of quaternary structure on protein conformational stability",
abstract = "ATP-dependent phosphoenolpyruvate (PEP) carboxykinases are found in plants and microorganisms, and catalyse the reversible formation of PEP, ADP, and CO2 from oxaloacetate plus ATP. These enzymes vary in quaternary structure although there is significant sequence identity among the proteins isolated from different sources. To help understand the influence of quaternary structure in protein stability, the urea-induced unfolding of free- and substrate-bound tetrameric Saccharomyces cerevisiae PEP carboxykinase is described and compared with the unfolding characteristics of the monomeric Escherichia coli enzyme [Eur. J. Biochem. 255 (1998) 439]. The urea-induced denaturation of S. cerevisiae PEP carboxykinase was studied by monitoring the enzyme activity, intrinsic protein fluorescence, circular dichroism (CD) spectra, and 1-anilino-8-naphthalenesulfonate (ANS) binding. The unfolding profiles were multi-steps, and formation of hydrophobic structures were detected. The data indicate that unfolding and dissociation of the enzyme tetramer are simultaneous events. Ligand binding, most notably PEP in the presence of MnCl2, conferred a marked protection against urea-induced denaturation. A similar protection effect was found when N-iodoacetyl-N′-(5-sulfo-1-napthyl)ethylene diamine (1,5-I-AEDANS) was covalently bound at Cys365, within the active site region. Refolding experiments indicated that total recovery of tertiary structure was only obtained from samples previously unfolded to less than 30{\%}. In the presence of substrates, complete refolding was achieved from samples originally denatured up to 50{\%}. The unfolding behaviour of S. cerevisiae PEP carboxykinase was found to be similar to that of E. coli PEP carboxykinase, however all steps take place at lower urea concentrations. These findings show that, at least for monomeric and tetrameric ATP-dependent PEP carboxykinases, quaternary structure does not contribute to protein conformational stability.",
keywords = "Ligand effects on unfolding, Phosphoenolpyruvate carboxykinase, Saccharomyces cerevisiae, Urea unfolding",
author = "Encinas, {M. Victoria} and Gonz{\'a}lez-Nilo, {Fernando D.} and Andreu, {Jos{\'e} M.} and Carlos Alfonso and Emilio Cardemil",
year = "2002",
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journal = "International Journal of Biochemistry and Cell Biology",
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TY - JOUR

T1 - Urea-induced unfolding studies of free- and ligand-bound tetrameric ATP-dependent Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

T2 - Influence of quaternary structure on protein conformational stability

AU - Encinas, M. Victoria

AU - González-Nilo, Fernando D.

AU - Andreu, José M.

AU - Alfonso, Carlos

AU - Cardemil, Emilio

PY - 2002/4/13

Y1 - 2002/4/13

N2 - ATP-dependent phosphoenolpyruvate (PEP) carboxykinases are found in plants and microorganisms, and catalyse the reversible formation of PEP, ADP, and CO2 from oxaloacetate plus ATP. These enzymes vary in quaternary structure although there is significant sequence identity among the proteins isolated from different sources. To help understand the influence of quaternary structure in protein stability, the urea-induced unfolding of free- and substrate-bound tetrameric Saccharomyces cerevisiae PEP carboxykinase is described and compared with the unfolding characteristics of the monomeric Escherichia coli enzyme [Eur. J. Biochem. 255 (1998) 439]. The urea-induced denaturation of S. cerevisiae PEP carboxykinase was studied by monitoring the enzyme activity, intrinsic protein fluorescence, circular dichroism (CD) spectra, and 1-anilino-8-naphthalenesulfonate (ANS) binding. The unfolding profiles were multi-steps, and formation of hydrophobic structures were detected. The data indicate that unfolding and dissociation of the enzyme tetramer are simultaneous events. Ligand binding, most notably PEP in the presence of MnCl2, conferred a marked protection against urea-induced denaturation. A similar protection effect was found when N-iodoacetyl-N′-(5-sulfo-1-napthyl)ethylene diamine (1,5-I-AEDANS) was covalently bound at Cys365, within the active site region. Refolding experiments indicated that total recovery of tertiary structure was only obtained from samples previously unfolded to less than 30%. In the presence of substrates, complete refolding was achieved from samples originally denatured up to 50%. The unfolding behaviour of S. cerevisiae PEP carboxykinase was found to be similar to that of E. coli PEP carboxykinase, however all steps take place at lower urea concentrations. These findings show that, at least for monomeric and tetrameric ATP-dependent PEP carboxykinases, quaternary structure does not contribute to protein conformational stability.

AB - ATP-dependent phosphoenolpyruvate (PEP) carboxykinases are found in plants and microorganisms, and catalyse the reversible formation of PEP, ADP, and CO2 from oxaloacetate plus ATP. These enzymes vary in quaternary structure although there is significant sequence identity among the proteins isolated from different sources. To help understand the influence of quaternary structure in protein stability, the urea-induced unfolding of free- and substrate-bound tetrameric Saccharomyces cerevisiae PEP carboxykinase is described and compared with the unfolding characteristics of the monomeric Escherichia coli enzyme [Eur. J. Biochem. 255 (1998) 439]. The urea-induced denaturation of S. cerevisiae PEP carboxykinase was studied by monitoring the enzyme activity, intrinsic protein fluorescence, circular dichroism (CD) spectra, and 1-anilino-8-naphthalenesulfonate (ANS) binding. The unfolding profiles were multi-steps, and formation of hydrophobic structures were detected. The data indicate that unfolding and dissociation of the enzyme tetramer are simultaneous events. Ligand binding, most notably PEP in the presence of MnCl2, conferred a marked protection against urea-induced denaturation. A similar protection effect was found when N-iodoacetyl-N′-(5-sulfo-1-napthyl)ethylene diamine (1,5-I-AEDANS) was covalently bound at Cys365, within the active site region. Refolding experiments indicated that total recovery of tertiary structure was only obtained from samples previously unfolded to less than 30%. In the presence of substrates, complete refolding was achieved from samples originally denatured up to 50%. The unfolding behaviour of S. cerevisiae PEP carboxykinase was found to be similar to that of E. coli PEP carboxykinase, however all steps take place at lower urea concentrations. These findings show that, at least for monomeric and tetrameric ATP-dependent PEP carboxykinases, quaternary structure does not contribute to protein conformational stability.

KW - Ligand effects on unfolding

KW - Phosphoenolpyruvate carboxykinase

KW - Saccharomyces cerevisiae

KW - Urea unfolding

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U2 - 10.1016/S1357-2725(01)00175-3

DO - 10.1016/S1357-2725(01)00175-3

M3 - Article

C2 - 11943595

AN - SCOPUS:0036220872

VL - 34

SP - 645

EP - 656

JO - International Journal of Biochemistry and Cell Biology

JF - International Journal of Biochemistry and Cell Biology

SN - 1357-2725

IS - 6

ER -