Phosphoenolpyruvate Carboxylase of Escherichia coli

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Abstract
Chemical modification of phosphoenolpyruvate carboxylase [EC 4.1.1.31] of Escherichia coli W with 2,3-butanedione in borate buffer resulted in inactivation of the enzyme. The reaction proceeded following pseudo-first-order kinetics, showing a maximal rate at pH 8.5. This inactivation was reversed when excess butanedione and borate were removed. DL-Phospho-lactate, a structural analog of the substrate, effectively protected the enzyme against inactivation. Concomitant with the butanedione modification, the enzyme was completely desensitized to fructose 1,6-bisphosphate and GTP, allosteric activators of the enzyme. Desensitization also occurred to acetyl coenzyme A, another allostenc activator and to L-aspartate, an allosteric inhibitor, to a lesser extent. Desensitization to these effectors occurred upon modification both in the presence and absence of DL-phospholactate. After the butanedione modification, the pH optimum for the activity and the Km value for the substrate remained unchanged, indicating that the desensitization to the effectors was not due to alteration of these kinetic properties. Amino acid analysis showed that 7 out of 49 arginyl residues per subunit were modified concomitant with a complete loss of activity. Modification of other amino acids was not detected under the experimental conditions used. Examination of the protective effect of DL-phospholactate against the butanedione inactivation and the order of reaction suggested that one arginyl residue is essential for the catalytic activity of the enzyme. Similarly, modification of the enzyme in the presence of fructose 1,6-bisphosphate and in its absence suggested that at least one, possibly two, arginyl residues are essential for the regulatory interaction with fructose 1,6-bisphosphate.