Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 1985 Mar;77(3):667–675. doi: 10.1104/pp.77.3.667

Diurnal Regulation of Phosphoenolpyruvate Carboxylase from Crassula1

Min-Xian Wu 1,2, Randolph T Wedding 1
PMCID: PMC1064583  PMID: 16664117

Abstract

Phosphoenolpyruvate carboxylase appears to be located in or associated with the chloroplasts of Crassula. As has been found with this enzyme in other CAM plants, a crude extract of leaves gathered during darkness and rapidly assayed for phosphoenolpyruvate carboxylase (PEPc) activity is relatively insensitive to inhibition by malate. After illumination begins, the PEPc activity becomes progressively more sensitive to malate. This enzyme also shows a diurnal change in activation by glucose-6-phosphate, with the enzyme from dark leaves more strongly activated than that from leaves in the light.

When the enzyme is partially purified in the presence of malate, the characteristic sensitivity of the day leaf enzyme is largely retained. Partial purification of the enzyme from dark leaves results in a small increase in sensitivity to malate inhibition.

Partially purified enzyme is found by polyacrylamide gel electrophoresis analysis to have two bands of PEPc activity. In enzymes from dark leaves, the slower moving band predominates, but in the light, the faster moving band is preponderant. Both of these bands are shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be composed of the same subunit of 103,000 daltons.

The enzyme partially purified from night leaves has a pH optimum of 5.6, and is relatively insensitive to malate inhibition over the range from pH 4.5 to 8. The enzyme from day leaves has a pH optimum of 6.6 and is strongly inhibited by malate at pH values below 7, but becomes insensitive at higher pH values.

Gel filtration of partially purified PEPc showed two activity peaks, one corresponding approximately to a dimer of the single subunit, and the other twice as large. The larger protein was relatively insensitive to malate inhibition, the smaller was strongly inhibited by malate.

Kinetic studies showed that malate is a mixed type inhibitor of the sensitive, day, enzyme, increasing Km for phosphoenolpyruvate and reducing Vmax. With the insensitive, night, enzyme, malate is a K type inhibitor, reducing the Km for phosphoenolpyruvate, but having little effect on Vmax. The inhibition of the insensitive enzyme by malate appears to be hysteretic, taking several minutes to be expressed during assay, probably indicating a change in the conformation or aggregation state of the enzyme.

Activation by glucose-6-phosphate is of the mixed type for the day form of the enzyme, causing both a decreased Km for phosphoenolpyruvate and an increased Vmax, but the night, or insensitive, form shows only an increase in Vmax in response to glucose-6-phosphate.

Full text

PDF
667

Images in this article

671Fig. 4
on p.671

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Buser-Suter C., Wiemken A., Matile P. A malic Acid permease in isolated vacuoles of a crassulacean Acid metabolism plant. Plant Physiol. 1982 Feb;69(2):456–459. doi: 10.1104/pp.69.2.456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Daniel P. P., Bryant J. A., Woodward F. I. Phosphoenolpyruvate carboxylase from pennywort (Umbilicus rupestris). Changes in properties after exposure to water stress. Biochem J. 1984 Mar 1;218(2):387–393. doi: 10.1042/bj2180387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Frieden C. Protein-protein interaction and enzymatic activity. Annu Rev Biochem. 1971;40:653–696. doi: 10.1146/annurev.bi.40.070171.003253. [DOI] [PubMed] [Google Scholar]
  4. Jones R., Wilkins M. B., Coggins J. R., Fewson C. A., Malcolm A. D. Phosphoenolpyruvate carboxylase from the crassulacean plant Bryophyllum fedtschenkoi Hamet et Perrier. Purification, molecular and kinetic properties. Biochem J. 1978 Nov 1;175(2):391–406. doi: 10.1042/bj1750391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Karn R. C., Kivic P. A., Hudock G. A. A procedure for the electrophoretic analysis of phosphoenolpyruvate carboxylase. Biochim Biophys Acta. 1973 Feb 15;293(2):567–569. doi: 10.1016/0005-2744(73)90366-5. [DOI] [PubMed] [Google Scholar]
  6. Kenyon W. H., Holaday A. S., Black C. C. Diurnal Changes in Metabolite Levels and Crassulacean Acid Metabolism in Kalanchoë daigremontiana Leaves. Plant Physiol. 1981 Nov;68(5):1002–1007. doi: 10.1104/pp.68.5.1002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Spalding M. H., Schmitt M. R., Ku S. B., Edwards G. E. Intracellular Localization of Some Key Enzymes of Crassulacean Acid Metabolism in Sedum praealtum. Plant Physiol. 1979 Apr;63(4):738–743. doi: 10.1104/pp.63.4.738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Wedding R. T., Black M. K. Physical and Kinetic Properties and Regulation of the NAD Malic Enzyme Purified from Leaves of Crassula argentea. Plant Physiol. 1983 Aug;72(4):1021–1028. doi: 10.1104/pp.72.4.1021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Winter K. Day/Night Changes in the Sensitivity of Phosphoenolpyruvate Carboxylase to Malate during Crassulacean Acid Metabolism. Plant Physiol. 1980 May;65(5):792–796. doi: 10.1104/pp.65.5.792. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES