title: Activity of maize leaf phosphoenolpyruvate carboxylase in relation to tautomerization and nonenzymatic decarboxylation of oxaloacetate.
abstract: The keto form of oxaloacetate (OAA), a product of phosphoenolpyruvate carboxylase (PEPC) activity, can undergo various nonenzymatic conversions which make conventional methods of assaying the enzyme difficult, because the products may either act as inhibitors or go undetected. In studies with PEPC isolated from leaves of maize, an assay coupled with reduction of OAA to malate was compared with product analysis using high-performance liquid chromatography and an assay based on Pi release. The results show that activity of the enzyme in the assay coupled to malate dehydrogenase is underestimated, to varying extents, depending on magnesium concentration, buffer, and pH. In the assay coupled to malate dehydrogenase, inaccuracies occur due to conversion of the keto form of OAA to the enol form, which is not utilized as a substrate, and due to loss of OAA by decarboxylation to pyruvate. The assay based on Pi formation is considered to give the true rate of catalysis. With this assay the pH optimum is 7.8, compared to 8.3-8.5 for the assay coupled to malate dehydrogenase. The metal enol complex of oxaloacetate (M-OAAenol) is an inhibitor of PEPC and conditions which are favorable for forming this tautomer, high pH with divalent metal ions or high concentrations of Tris buffer at a pH below its pKa value, limit catalysis. Glycine stimulates enzyme activity, and it may have its effect by preventing the formation of the hydrated M-OAAenol complex and maintaining more of the OAA in the keto form. This interpretation is consistent with glycine stimulation of malate synthesis in the assay of PEPC coupled to malate dehydrogenase, with glycine stimulation of the decarboxylation of OAA, and with a reduction in the level of the M-OAAenol complex in the presence of glycine.
mesh_terms: D002021:Buffers; D002262:Carboxy-Lyases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005998:Glycine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D008274:Magnesium / Q000494:pharmacology; D008291:Malate Dehydrogenase / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010071:Oxaloacetates / Q000378:metabolism*; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism; D003313:Zea mays
chemical_list: D002021:Buffers; D010071:Oxaloacetates; D011773:Pyruvates; D009243:NAD; D008291:Malate Dehydrogenase; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase; D008274:Magnesium; D005998:Glycine