Inactivation of Maize Phosphoenolpyruvate Carboxylase by Urea

Abstract

Phosphoenolpyruvate carboxylase purified from leaves of maize (Zea mays, L.) is sensitive to the presence of urea. Exposure to 2.5 M urea for 30 min completely inactivates the enzyme, whereas for a concentration of 1.5 M urea, about 1 h is required. Malate appears to have no effect on inactivation by urea of phosphoenolpyruvate carboxylase. However, the presence of 20 mm phosphoenolpyruvate or 20 mm glucose-6-phosphate prevents significant inactivation by 1.5 M urea for at least 1 h. The inactivation by urea is reversible by dilution. The inhibition by urea and the protective effects of phosphoenolpyruvate and glucose-6-phosphate are associated with changes in aggregation state.Although the effects of urea on proteins are varied and subtle (13), it is commonly considered that a major aspect of action of urea is to induce the dissociation of oligomeric proteins (3,5,6,12). Although some enzymes have been found (6) to give varied responses depending on the concentration of urea applied, urea is widely used to dissociate large enzyme molecules. In some instances, urea in concentrations from 3 to 8 M has been shown to dissociate tetrameric proteins to their subunits. This general pattern is shown by fumarase (5), glutamate dehydrogenase (1), and phosphofructokinase (6), in which the active tetramer is dissociated to monomers by the presence of urea. This effect of urea, which disaggregates proteins by breaking hydrogen bonds (12), offers a different way of probing the factors involved in equilibria of enzymic oligomers. Our interest in this question arises from the aggregation and disaggregation of PEPC,2 which is a major factor in regulating the activity of that enzyme (15,17).We have studied the effect of moderate concentrations of urea on the inactivation of PEPC and have investigated the effect on the response to urea of ligands known to regulate the enzyme. We have also shown, using light scattering, that the effect of urea is to dissociate the enzyme. MATERIALS AND METHODS Chemicals and EnzymesThe trisodium salt of PEP and the disodium salt of glc-6-P were obtained from Sigma. Malate was from Aldrich and l Supported in part by grant DCB 88-12484 from the National Science Foundation.2 Abbreviations: PEPC, phosphoenolpyruvate carboxylase; PEP, phosphoenolpyruvate; glc-6-P, glucose-6-phosphate; U, unit of enzyme activity, micromoles per minute per milligram of protein. 1366NADH from Boehringer-Mannheim. All chemicals were of the highest quality commercially available. Malate dehydrogenase (from pig heart) and lactate dehydrogenase (from hog muscle), both suspended in 50% glycerol, were purchased from Boehringer-Mannheim.Enzyme PEPC was purified from the deribbed leaves of 3-to 4-week-old seedlings of maize by the method previously described (9). At the time of harvest, the leaves had been exposed to sunlight for 4 to 6 h in a greenhouse maintained at a daytime temperature of 300C. The specific activity at the time of purification was 22 U/mg of protein.The activity of PEPC was determined using 1-mL assays...