Effects of High Pressure on the Stability and Activity of Allosteric Phosphoenolpyruvate Carboxylase from Escherichia coli*

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Abstract
Effects of hydrostatic pressure up to 3, 000 atm on phosphoenolpyruvate (PEP) carbo-xylase [EC 4.1.1.31] were studied. The enzyme in the absence of its ligands lost some activity by the compression at 1, 000 atm for 15 min at 22°C and was inactivated completely by the compression at 2, 000 atm for 15 min in complete nonreversibility upon release of pressure. An activation volume for the inactivation was evaluated as about −120 ml/mole of the enzyme based on the dependency of inactivation rate on the magnitude of pressure. Investigation of the effect of each ligand on the pressure inactivation of the enzyme revealed the following facts: (i) PEP, one of the substrates, and the allosteric activators such as acetyl-CoA and fructose 1, 6-di-phosphate showed no effect; (ii) L-Aspartate, one of the allosteric inhibitors, showed a marked protective effect. The extent of protection increased with increasing concentrations of L-aspartate and was saturated at 10 mM; (iii) Long chain fatty acid such as laurate, the other allosteric activator, markedly accelerated the pressure inactivation. Discussion is made on the conformational states induced by each ligand based on these results. In addition, the response of enzyme to the effectors at 500 atm where the native subunit structure was possibly retained, was investigated with the intension of getting some information on the change in partial molar volume of the enzyme associated with the allosteric transition. Although no significant pressure effect was observed in this regard, a possible usefulness of this approach is discussed.

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