Abstract
Initial velocity studies of rat liver cytosolic P-enolpyruvate carboxykinase in the direction of P-enolpyruvate formation gave intersecting double reciprocal plots indicating that the reaction conforms to a sequential reaction pathway. A complete product inhibition study with MnGDP-, P-enolpyruvate, and HCO3- as product inhibitors indicated that all patterns were noncompetitive. Isotope exchange at equilibrium with exchange between the substrate/product pairs GTP/GDP, oxalacetate/HCO3-, and oxalacetate/P-enolpyruvate while varying the concentration of substrate/product pairs in fixed constant ratio gave no complete inhibitory patterns as the concentration of the constant ratio pairs approached saturation. The exchange rates between the substrate/product pairs differed by a factor of 40 when compared under the same assay conditions. These results were interpreted in terms of a random reaction mechanism in which true dead-end complexes do not form and in which the rate-limiting step is not the interconversion of the ternary quaternary central complexes. In addition to the formation of P-enolpyruvate from oxalacetate and MnGTP2-, the enzyme catalyzes the decarboxylation of oxalacetate to pyruvate in the absence of MnGTP2-. This reaction occurs only slowly in the absence of GDP and most rapidly in the presence of MnGDP-. When only MnGTP2- and oxalacetate are present, no pyruvate is formed, and oxalacetate is converted stoichiometrically to P-enolpyruvate. The enzyme also catalyzes the exchange of [14C]GDP into GTP in the absence of P-enolpyruvate. This exchange is stimulated by the presence of HCO3-. When enzyme is incubated with MnGTP2- in the presence or absence of HCO3-, there is no hydrolysis to form GDP and P(i). The two partial reactions, namely the exchange of [14C]GDP with the E. HC MnGTP or E. MnGTP complex and the formation of pyruvate from the E. oxalacetate MnGDP complex provide pathways by which the expected dead-end complexes can be converted to enzyme forms which can return to the catalytic or exchange sequence.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3648-3659 |
| Number of pages | 12 |
| Journal | Journal of Biological Chemistry |
| Volume | 253 |
| Issue number | 10 |
| State | Published - 1978 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Kinetic mechanism of phosphoenolpyruvate carboxykinase (GTP) from rat liver cytosol. Product inhibition, isotope exchange at equilibrium, and partial reactions. / Jomain-Baum, M.; Schramm, Vern L.
In: Journal of Biological Chemistry, Vol. 253, No. 10, 1978, p. 3648-3659.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Kinetic mechanism of phosphoenolpyruvate carboxykinase (GTP) from rat liver cytosol. Product inhibition, isotope exchange at equilibrium, and partial reactions
AU - Jomain-Baum, M.
AU - Schramm, Vern L.
PY - 1978
Y1 - 1978
N2 - Initial velocity studies of rat liver cytosolic P-enolpyruvate carboxykinase in the direction of P-enolpyruvate formation gave intersecting double reciprocal plots indicating that the reaction conforms to a sequential reaction pathway. A complete product inhibition study with MnGDP-, P-enolpyruvate, and HCO3- as product inhibitors indicated that all patterns were noncompetitive. Isotope exchange at equilibrium with exchange between the substrate/product pairs GTP/GDP, oxalacetate/HCO3-, and oxalacetate/P-enolpyruvate while varying the concentration of substrate/product pairs in fixed constant ratio gave no complete inhibitory patterns as the concentration of the constant ratio pairs approached saturation. The exchange rates between the substrate/product pairs differed by a factor of 40 when compared under the same assay conditions. These results were interpreted in terms of a random reaction mechanism in which true dead-end complexes do not form and in which the rate-limiting step is not the interconversion of the ternary quaternary central complexes. In addition to the formation of P-enolpyruvate from oxalacetate and MnGTP2-, the enzyme catalyzes the decarboxylation of oxalacetate to pyruvate in the absence of MnGTP2-. This reaction occurs only slowly in the absence of GDP and most rapidly in the presence of MnGDP-. When only MnGTP2- and oxalacetate are present, no pyruvate is formed, and oxalacetate is converted stoichiometrically to P-enolpyruvate. The enzyme also catalyzes the exchange of [14C]GDP into GTP in the absence of P-enolpyruvate. This exchange is stimulated by the presence of HCO3-. When enzyme is incubated with MnGTP2- in the presence or absence of HCO3-, there is no hydrolysis to form GDP and P(i). The two partial reactions, namely the exchange of [14C]GDP with the E. HC MnGTP or E. MnGTP complex and the formation of pyruvate from the E. oxalacetate MnGDP complex provide pathways by which the expected dead-end complexes can be converted to enzyme forms which can return to the catalytic or exchange sequence.
AB - Initial velocity studies of rat liver cytosolic P-enolpyruvate carboxykinase in the direction of P-enolpyruvate formation gave intersecting double reciprocal plots indicating that the reaction conforms to a sequential reaction pathway. A complete product inhibition study with MnGDP-, P-enolpyruvate, and HCO3- as product inhibitors indicated that all patterns were noncompetitive. Isotope exchange at equilibrium with exchange between the substrate/product pairs GTP/GDP, oxalacetate/HCO3-, and oxalacetate/P-enolpyruvate while varying the concentration of substrate/product pairs in fixed constant ratio gave no complete inhibitory patterns as the concentration of the constant ratio pairs approached saturation. The exchange rates between the substrate/product pairs differed by a factor of 40 when compared under the same assay conditions. These results were interpreted in terms of a random reaction mechanism in which true dead-end complexes do not form and in which the rate-limiting step is not the interconversion of the ternary quaternary central complexes. In addition to the formation of P-enolpyruvate from oxalacetate and MnGTP2-, the enzyme catalyzes the decarboxylation of oxalacetate to pyruvate in the absence of MnGTP2-. This reaction occurs only slowly in the absence of GDP and most rapidly in the presence of MnGDP-. When only MnGTP2- and oxalacetate are present, no pyruvate is formed, and oxalacetate is converted stoichiometrically to P-enolpyruvate. The enzyme also catalyzes the exchange of [14C]GDP into GTP in the absence of P-enolpyruvate. This exchange is stimulated by the presence of HCO3-. When enzyme is incubated with MnGTP2- in the presence or absence of HCO3-, there is no hydrolysis to form GDP and P(i). The two partial reactions, namely the exchange of [14C]GDP with the E. HC MnGTP or E. MnGTP complex and the formation of pyruvate from the E. oxalacetate MnGDP complex provide pathways by which the expected dead-end complexes can be converted to enzyme forms which can return to the catalytic or exchange sequence.
UR - http://www.scopus.com/inward/record.url?scp=0017822596&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0017822596&partnerID=8YFLogxK
M3 - Article
C2 - 649593
AN - SCOPUS:0017822596
VL - 253
SP - 3648
EP - 3659
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 10
ER -