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. 1970 Nov;120(1):79–93. doi: 10.1042/bj1200079

Some aspects of the kinetics of rat liver pyruvate carboxylase

Janet M Wimhurst 1, K L Manchester 1
PMCID: PMC1179571  PMID: 4321933

Abstract

1. The kinetics of rat liver pyruvate carboxylase were examined and the effect of various agents as activators or inhibitors determined. 2. Essentially similar results were obtained in comparisons of kinetics determined by a radioactivity method involving extracts of acetone-dried powders from whole livers and with a spectrophotometric assay using partially purified enzyme from the mitochondrial fraction. Activity per g of liver from fed or starved rats assayed under optimum substrate and activator conditions was 3 or 6 μmol of oxaloacetate formed/min at 30°C, respectively. 3. The enzyme exhibited cold-lability and lost activity on standing, even in 1.5m-sucrose. 4. The Km towards pyruvate was about 0.33mm and towards bicarbonate 4.2mm. Km towards MgATP2− was 0.14mm. Mg2+ ions activated the enzyme, in addition to their role in MgATP2− formation. From calculations of likely concentrations of free Mg2+ in the assay medium a Ka towards Mg2+ of about 0.25mm was deduced. Mn2+ also activated the enzyme as well as Mg2+, but at much lower concentrations. It appeared to be inhibitory when concentrations of free Mn2+ as low as 0.1mm were present. 5. Excess of ATP is inhibitory, and this appears at least in part independent of the trapping of Mg2+. 6. Both Co2+ and Zn2+ were inhibitory; 2mol of the latter appeared to be bound even in the presence of excess of Mg2+ and the inhibition was time-dependent. 7. Ca2+ inhibited by competition with Mg2+ (Ki about 0.38mm). The inhibition due to Ca2+ was less pronounced when activation was with Mn2+. Inhibition by Ca2+ and ATP appeared to be additive. 8. Hill plots suggested that no interactions occurred between ATP-binding sites. Although similar plots for total Mg2+ gave n=3.6, no conclusions could be drawn due to the chelation of the cation with other components of the assay. Similar difficulties arose in assessing the values for Ca2+. 9. The enzyme was inactive in the absence of acetyl-CoA and showed a sigmoidal response in its presence. Ka was about 0.1mm with possibly up to four binding sites. Malonyl-CoA was a competitive inhibitor, with Ki 0.01mm. 10. There was no apparent inhibition by glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, acetoacetate, β-hydroxybutyrate, malate, aspartate, pyruvate, palmitoylcarnitine, octanoate, glutathione, butacaine, triethyltin or potassium chloride under the conditions used. Inhibition was found with citrate (possibly by chelation) and adenosine, and also by phosphoenolpyruvate, AMP, ADP and cyclic AMP, Ki towards the last four being 0.55, 0.76, 0.25 and 1.4mm respectively.

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Selected References

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