Resumen
Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is completely inactivated by the 2′,3′-dialdehyde derivative of ATP (oATP) in the presence of Mn2+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 ± 17 μM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30 ° C is 0.200 ± 0.045 min-1. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unaltered Km but a decreased V as compared with native phosphoenolpyruvate carboxykinase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pKa of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8-14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase.
| Idioma original | English |
|---|---|
| Páginas (desde-hasta) | 38-45 |
| Número de páginas | 8 |
| Publicación | Archives of Biochemistry and Biophysics |
| Volumen | 267 |
| N.º | 1 |
| DOI | |
| Estado | Published - 15 nov 1988 |
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ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
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Affinity labeling of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase with the 2′,3′-dialdehyde derivative of ATP. / Saavedra, Claudia; Araneda, Sandra; Cardemil, Emilio.
En: Archives of Biochemistry and Biophysics, Vol. 267, N.º 1, 15.11.1988, p. 38-45.Resultado de la investigación: Article
TY - JOUR
T1 - Affinity labeling of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase with the 2′,3′-dialdehyde derivative of ATP
AU - Saavedra, Claudia
AU - Araneda, Sandra
AU - Cardemil, Emilio
PY - 1988/11/15
Y1 - 1988/11/15
N2 - Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is completely inactivated by the 2′,3′-dialdehyde derivative of ATP (oATP) in the presence of Mn2+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 ± 17 μM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30 ° C is 0.200 ± 0.045 min-1. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unaltered Km but a decreased V as compared with native phosphoenolpyruvate carboxykinase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pKa of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8-14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase.
AB - Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is completely inactivated by the 2′,3′-dialdehyde derivative of ATP (oATP) in the presence of Mn2+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 ± 17 μM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30 ° C is 0.200 ± 0.045 min-1. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unaltered Km but a decreased V as compared with native phosphoenolpyruvate carboxykinase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pKa of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8-14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase.
UR - http://www.scopus.com/inward/record.url?scp=0024289219&partnerID=8YFLogxK
U2 - 10.1016/0003-9861(88)90005-7
DO - 10.1016/0003-9861(88)90005-7
M3 - Article
C2 - 3058040
AN - SCOPUS:0024289219
VL - 267
SP - 38
EP - 45
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
SN - 0003-9861
IS - 1
ER -