Effects of pH on inactivation of maize phosphoenolpyruvate carboxylase.
Maize leaf phosphoenolpyruvate carboxylase (PEPC) is inactivated by incubation at pH's above neutrality. Both the amount and the rapidity of inactivation increase as the pH rises. The presence of phosphoenolpyruvate (PEP), malate, glucose 6-phosphate and dithiothreitol in the incubation medium give protection to the enzyme. While the presence of PEP during incubation at pH 8 prevents inactivation, the level of PEP in the assay after incubation has no effect on the relative inactivation. When the enzyme is incubated at pH 7 with 5 mM malate (a treatment known to cause dimerization) subsequent assay at saturating levels of MgPEP completely restores activity while assay at less than Km MgPEP produces greater than 99% inhibition of the same sample, showing that high PEP concentration has reconverted the PEPC to the malate-resistant tetramer. Thus the protective effect of PEP against inactivation at high pH probably is not related to its effect on the aggregation state of the enzyme but rather is due to the presence of PEP at the active site. Protection of PEPC at pH 8 by EDTA and its inactivation by low concentrations of Cu2- indicates that the loss of activity at high pH probably is in a sense an artifact resulting from the binding to a deprotinated cysteine of heavy metal ions contaminating the enzyme preparation or present in reagents. This suggests that caution should be used in the interpretation of experiments involving PEPC activity at alkaline pH's.
Wedding
R T
RT
Department of Biochemistry, University of California, Riverside 92521.
Black
M K
MK
D003300:Copper / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008293:Malates / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000494:pharmacology*; D010730:Phosphoenolpyruvate Carboxylase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D003313:Zea mays / Q000201:enzymology*
D005958:Glucosephosphates; D008293:Malates; D013438:Sulfhydryl Compounds; D019298:Glucose-6-Phosphate; D010728:Phosphoenolpyruvate; D003300:Copper; D004492:Edetic Acid; D010730:Phosphoenolpyruvate Carboxylase