Pyruvate carboxylase from a thermophilic Bacillus. Studies on the specificity of activation by acyl derivatives of coenzyme A and on the properties of catalysis in the absence of activator

Abstract

1. Oxaloacetate synthesis catalysed by pyruvate carboxylase from a thermophilic Bacillus in the absence of acetyl-CoA required addition of high concentrations of pyruvate, MgATP(2-) and HCO(3) (-), and at 45 degrees C occurred at a maximum rate approx. 20% of that in the presence of a saturating concentration of acetyl-CoA. The apparent K(m) for HCO(3) (-) at pH7.8 was 400mm without acetyl-CoA, and 16mm with a saturating activator concentration. The relationship between reciprocal initial rate and reciprocal MgATP(2-) concentration was non-linear (convex-down) in the absence of acetyl-CoA, but the extent of deviation decreased as the activator concentration was increased. The relationship between reciprocal initial rate and reciprocal pyruvate concentration was non-linear (convex-down) in the presence or absence of acetyl-CoA. 2. The optimum pH for catalysis of oxaloacetate synthesis was similar in the presence or absence of acetyl-CoA. The variation with pH of apparent K(m) for HCO(3) (-) implicated residue(s) with pK(a) 8.6 in catalysis of the activator-independent oxaloacetate synthesis. 3. Linear Arrhenius and van't Hoff plots were observed for the temperature-dependence of oxaloacetate synthesis in the absence of acetyl-CoA over the range 25-55 degrees C. E(a) (activation energy) was 56.3kJ/mol and DeltaH(double dagger) (HCO(3) (-)) (enthalpy of activation) was -38.6kJ/mol. In the presence of acetyl-CoA, biphasic Arrhenius and van't Hoff plots are observed with a change of slope at 30 degrees C in each case. E(a) was 43.7 and 106.3kJ/mol above and below 30 degrees C respectively. 4. Incubation of Bacillus pyruvate carboxylase with trinitrobenzenesulphonate caused specific inactivation of acetyl-CoA-dependent catalytic activity associated with the incorporation of 1.3+/-0.2 trinitrophenyl residues per subunit. Activator-independent catalysis and regulatory inhibition by l-aspartate were unaffected. The rate of inactivation of acetyl-CoA-dependent catalysis by trinitrobenzenesulphonate was specifically decreased by addition of acetyl-CoA and other acetyl-CoA and other acyl-CoA species, but complete protection was not obtained. 5. All alkylacyl derivatives of CoA tested activated Bacillus pyruvate carboxylase; acetyl-CoA was the most effective. The apparent K(a) exhibited a biphasic relationship with acyl-chain length for the straight-chain homologues. Certain long-chain acyl-CoA species showed additional activation at a high concentration. Weak activation occurred on addition of CoA or adenosine 3',5'-bisphosphate, but carboxyacyl-CoA species and derivatives containing a modified phosphoadenosyl group were inhibitory. Thioesters of CoA with non-carboxylic acids, e.g. methanesulphonyl-CoA, serve as activators of the thermophilic Bacillus and Saccharomyces cerevisiae pyruvate carboxylases, but as inhibitors of pyruvate carboxylases obtained from chicken and rat liver. 6. alpha-Oxoglutarate mimics the effect of l-aspartate as a regulatory inhibitor of the pyruvate carboxylases from both the thermophilic Bacillus ...

Pyruvate carboxylase from Aspergillus nidulans

“…niduluns is only weakly activated by acyl derivatives of CoASH even in the presence of non-saturating concentrations of its substrates [4] and is subject to regulatory inhibition by Laspartate and 2-oxoglutarate [5]. These properties are generally similar to those described previously for pyruvate carboxylase from Succhuromyces cerevisiae [6,7] which has also been reported to have a cytosolic localisation [8]. Hence it has been suggested that the cytosolic and mitochondrial isoenzymes of pyruvate carboxylase may possess characteristically different regulatory properties [l 1.…”

Mechanisms of Inhibition of Rhizobium etli Pyruvate Carboxylase by l-Aspartate

“…Previously, IC 50 values for the inhibition by l -aspartate for PC have been reported to be in the range of 0.6–4 mM, 10,11,21 while Dunn et al 22 reported that 10 mM l -aspartate only marginally inhibited the enzyme from Sinorhizobium meliloti . Thus, the K i value of 1.3 mM obtained for RePC in this study is at the lower end of the range.…”

“…There is, however, some evidence to suggest that this is not the case. First, Libor et al 21 showed that modification of the thermophilic Bacillus PC with trinitrobenzenesulfonate specifically inhibited acetyl-CoA-dependent enzyme activity but had no effect on the acetyl-CoA-independent activity or its inhibition by l -aspartate. Moreover, the presence of acetyl-CoA protected the enzyme against modification.…”

Activation and Inhibition of Pyruvate Carboxylase from Rhizobium etli

“…For example, the apparent K m for HCO 3 − observed with PC from B. Thermodenitrificans (32) was 16 mM in the presence of acetyl-CoA and the absence of acetyl-CoA resulted in a 25-fold increase in the K m for HCO 3 − (400 mM). Similar effects were observed in Re PC where the K m for HCO 3 − in Re PC determined in the presence of acetyl-CoA was 10.8 ± 0.4 mM, comparable to those for other bacterial PCs (33).…”