Isotope trapping and positional isotope exchange with rat and chicken liver phosphoenolpyruvate carboxykinases.
Isotope-trapping studies of the enzyme.MgGTP complex were carried out with rat liver cytosolic and chicken liver mitochondrial phosphoenolpyruvate carboxykinases. For the rat liver enzyme, MgGTP was partially trapped from both E.MgGTP and E.MgGTP.OAA complexes, consistent with a steady-state random mechanism. For the chicken liver enzyme, MgGTP was 100% trapped from the E.MgGTP.OAA complex, consistent with a steady-state ordered mechanism. The rate constants for the interaction of MgGTP with the free enzymes are approximately 10(7) M-1 S-1, somewhat lower than the diffusion limit for association. The dissociation rate for the enzyme.MgGTP complexes is 26-92 s-1, reflecting a tightly bound complex with high commitment to catalysis in the presence of oxaloacetate. Positional isotope-exchange studies were also carried out with phosphoenolpyruvate carboxykinases from rat and chicken. No exchange if the beta gamma-18O in [beta gamma-18O, gamma-18O3]GTP to form [beta-18O, gamma-18O3]GTP was detected in the absence of oxaloacetate. In the presence of oxaloacetate, no positional isotope exchange of [beta gamma-18O, gamma-18O3]GTP was detected during initial rate conditions. The results indicate that at least one of the products dissociates rapidly from the E.MgGDP.PEP.CO2 complex relative to the net rate of MgGTP formation from the E.MgGDP.PEP.CO2 complex. A rapid equilibrium between the central complexes in which the beta-phosphoryl of GDP is restricted with respect to torsional rotation cannot be excluded but is unlikely on the basis of the relative rates of catalysis and torsional rotation. The addition of Mn2+, an activator of phosphoenolpyruvate carboxykinase, did not influence the positional isotope-exchange results.(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
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Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
Sato
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Schramm
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D000818:Animals; D002645:Chickens; D006160:Guanosine Triphosphate / Q000737:chemistry; D007554:Isotopes; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry* / Q000302:isolation & purification; D051381:Rats
D007554:Isotopes; D006160:Guanosine Triphosphate; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D008274:Magnesium