Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1992 Dec;100(4):2035–2040. doi: 10.1104/pp.100.4.2035

Evidence for the Existence of Two Essential and Proximal Cysteinyl Residues in NADP-Malic Enzyme from Maize Leaves 1

María F Drincovich 1, Claudia P Spampinato 1, Carlos S Andreo 1
PMCID: PMC1075903  PMID: 16653236

Abstract

Incubation of maize (Zea mays) leaf NADP-malic enzyme with monofunctional and bifunctional N-substituted maleimides results in an irreversible inactivation of the enzyme. Inactivation by the monofunctional reagents, N-ethylmaleimide (NEM) and N-phenylmaleimide, followed pseudo-first-order kinetics. The maximum inactivation rate constant for phenylmaleimide was 10-fold higher than that for NEM, suggesting a possible hydrophobic microenvironment of the residue(s) involved in the modification of the enzyme. In contrast, the inactivation kinetics with the bifunctional maleimides, ortho-, meta-, and para-phenylenebismaleimide, were biphasic, probably due to different reactivities of the groups reacting with the two heads of these bifunctional reagents, with a possible cross-linking of two sulfhydryl groups. The inactivation by mono and bifunctional maleimides was partially prevented by Mg2+ and l-malate, and NADP prevented the inactivation almost totally. Determination of the number of reactive sulfhydryl groups of the native enzyme with [3H]NEM in the absence or presence of NADP showed that inactivation occurred concomitantly with the modification of two cysteinyl residues per enzyme monomer. The presence of these two essential residues was confirmed by titration of sulfhydryl groups with [3H]NEM in the enzyme previously modified by o-phenylenebismaleimide in the absence or presence of NADP.

Full text

PDF
2035

Selected References

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

  1. Börsch D., Westhoff P. Primary structure of NADP-dependent malic enzyme in the dicotyledonous C4 plant Flaveria trinervia. FEBS Lett. 1990 Oct 29;273(1-2):111–115. doi: 10.1016/0014-5793(90)81063-t. [DOI] [PubMed] [Google Scholar]
  2. Chang G. G., Hsu R. Y. Mechanism of pigeon liver malic enzyme: kinetics, specificity, and half-site stoichiometry of the alkylation of a cysteinyl residue by the substrate-inhibitor bromopyruvate. Biochemistry. 1977 Jan 25;16(2):311–320. doi: 10.1021/bi00621a024. [DOI] [PubMed] [Google Scholar]
  3. Davies D. D., Patil K. D. Regulation of 'malic' enzyme of Solanum tuberosum by metabolites. Biochem J. 1974 Jan;137(1):45–53. doi: 10.1042/bj1370045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Edwards G. E., Andreo C. S. NADP-malic enzyme from plants. Phytochemistry. 1992 Jun;31(6):1845–1857. doi: 10.1016/0031-9422(92)80322-6. [DOI] [PubMed] [Google Scholar]
  5. Frenkel R. Regulation and physiological functions of malic enzymes. Curr Top Cell Regul. 1975;9:157–181. doi: 10.1016/b978-0-12-152809-6.50012-3. [DOI] [PubMed] [Google Scholar]
  6. Hsu R. Y. Pigeon liver malic enzyme. Mol Cell Biochem. 1982 Mar 5;43(1):3–26. doi: 10.1007/BF00229535. [DOI] [PubMed] [Google Scholar]
  7. Iglesias A. A., Andreo C. S. NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves. Kinetic properties of different oligomeric structures. Eur J Biochem. 1990 Sep 24;192(3):729–733. doi: 10.1111/j.1432-1033.1990.tb19283.x. [DOI] [PubMed] [Google Scholar]
  8. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  9. Magnuson M. A., Morioka H., Tecce M. F., Nikodem V. M. Coding nucleotide sequence of rat liver malic enzyme mRNA. J Biol Chem. 1986 Jan 25;261(3):1183–1186. [PubMed] [Google Scholar]
  10. Moroney J. V., Warncke K., McCarty R. E. The distance between thiol groups in the gamma subunit of coupling factor 1 influences the proton permeability of thylakoid membranes. J Bioenerg Biomembr. 1982 Dec;14(5-6):347–359. doi: 10.1007/BF00743063. [DOI] [PubMed] [Google Scholar]
  11. Penefsky H. S. Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase. J Biol Chem. 1977 May 10;252(9):2891–2899. [PubMed] [Google Scholar]
  12. Pry T. A., Hsu R. Y. Mechanism of pigeon liver malic enzyme. Reactivity of class II sulfhydryl groups as a conformational probe for the "half-of-the-sites" reactivity of the enzyme with bromopyruvate. Biochemistry. 1978 Sep 19;17(19):4024–4029. doi: 10.1021/bi00612a023. [DOI] [PubMed] [Google Scholar]
  13. Rothermel B. A., Nelson T. Primary structure of the maize NADP-dependent malic enzyme. J Biol Chem. 1989 Nov 25;264(33):19587–19592. [PubMed] [Google Scholar]
  14. Scrutton M. C., Utter M. F. Pyruvate carboxylase. V. Interaction of the enzyme with adenosine triphosphate. J Biol Chem. 1965 Oct;240(10):3714–3723. [PubMed] [Google Scholar]
  15. Tang C. L., Hsu R. Y. Mechanism of pigeon liver malic enzyme. Modification of sulfhydryl groups by 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide. J Biol Chem. 1974 Jun 25;249(12):3916–3922. [PubMed] [Google Scholar]
  16. Weiss M. A., McCarty R. E. Cross-linking within a subunit of coupling factor 1 increases the proton permeability of spinach chloroplast thylakoids. J Biol Chem. 1977 Nov 25;252(22):8007–8012. [PubMed] [Google Scholar]
  17. Wells J. A., Yount R. G. Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment 1. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4966–4970. doi: 10.1073/pnas.76.10.4966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Willeford K. O., Wedding R. T. pH Effects on the Activity and Regulation of the NAD Malic Enzyme. Plant Physiol. 1987 Aug;84(4):1084–1087. doi: 10.1104/pp.84.4.1084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wise L. S., Rubin C. S. Rapid purification and radioimmunoassay of cytosolic malic enzyme. Anal Biochem. 1984 Jul;140(1):256–263. doi: 10.1016/0003-2697(84)90162-3. [DOI] [PubMed] [Google Scholar]

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

RESOURCES