Pyruvate Carboxylase from Aspergillus nidulans
Authors: OSMANI Steven A. , MARSTON Fiona A. O. , SELMES Ian P. , CHAPMAN Astrid G. , SCRUTTON Michael C. 
Published: July 2010

Partially purified preparations of pyruvate carboxylase from Aspergillus nidulans are inhibited significantly only by l‐aspartate and 2‐oxoglutarate out of nine dicarboxylic and tricarboxylic acids tested. Initial velocity/inhibitor concentration relationships are sigmoid for both dicarboxylic acids and, for l‐aspartate, inhibition is incomplete even at saturating concentrations.

l‐Aspartate acts as a competitive inhibitor with respect to HCO3‐ and a non‐competitive inhibitor with respect to pyruvate and MgATP2−. Secondary slope and intercept plots are non‐linear functions of l‐aspartate concentration.

2‐Oxoglutarate induces complex kinetic behaviour for all three substrates. Inhibition appears competitive with respect to pyruvate and at lower substrate concentrations uncompetitive with respect to HCO3−. Both the sigmoid character of the initial velocity/[2‐oxoglutarate] relationship and the concentration of 2‐oxoglutarate required for 50% inhibition ([I]0.5) are markedly and specifically increased by an increase in pyruvate concentration.

Addition of acetyl‐CoA causes activation in the presence of either l‐aspartate or 2‐oxoglutarate but does not activate the enzyme in the absence of these dicarboxylic acids. In the presence of 2‐oxoglutarate this effect is less marked at more alkaline pH. Longer‐chain homologues of acetyl‐CoA also activate under these conditions with oleoyl‐CoA as the most effective activator.

An apparently competitive relationship exists between the effects of acetyl‐CoA and l‐aspartate on catalysis by pyruvate carboxylase from A. nidulans.l‐Aspartate increases the apparent KA for acetyl‐CoA but does not change the hyperbolic character of the initial velocity/[acetyl‐CoA] relationship. Acetyl‐CoA has no effect on the maximal extent of inhibition by l‐aspartate but increases both the sigmoidicity of the initial velocity [l‐aspartate] relationship and the concentration of l‐aspartate giving 50% inhibition.

A non‐competitive relationship exists between the effects of acetyl‐CoA and 2‐oxoglutarate. The relationship between initial velocity and [acetyl‐CoA] remains hyperbolic in the presence of 2‐oxoglutarate but the apparent KA is increased. Acetyl‐CoA causes a slight increase in the concentration of 2 required for 50% inhibition but decreases the sigmoid character of the initial velocity/[2‐oxoglutarate] relationship.

Incubation of pyruvate carboxylase from A. nidulans with trinitrobenzenesulphonate causes selective loss of the capacity for activation by acetyl‐CoA with no significant effect on the extent of inhibition by l‐aspartate or 2‐oxoglutarate. Catalytic activity in the absence of these effectors decreases less markedly. These effects are largely prevented if the enzyme is incubated with trinitrobenzenesulphonate in the presence of acetyl‐CoA.

We propose that pyruvate carboxylase from A. nidulans carries an allosteric acyl‐CoA activator site but that under the conditions examined occupancy of this site affects catalysis only if either l‐aspartate or 2‐oxoglutarate is present. We further propose that the enzyme carries distinct regulatory sites for l‐aspartate and 2‐oxoglutarate.