Some aspects of the kinetics of rat liver pyruvate carboxylase

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 K_m towards pyruvate was about 0.33mm and towards bicarbonate 4.2mm. K_m 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 K_a 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⁺ (K_i 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. K_a was about 0.1mm with possibly up to four binding sites. Malonyl-CoA was a competitive inhibitor, with K_i 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, K_i towards the last four being 0.55, 0.76, 0.25 and 1.4mm respectively.