Primary structure and analysis of the location of the regulatory disulfide bond of pea chloroplast NADP-malate dehydrogenase.
Purified pea chloroplast NADP-malate dehydrogenase (S)-malate: NADP+ oxidoreductase, EC 1.1.1.82) was digested with trypsin and the resulting peptides were separated by HPLC and sequenced. Together with the information from earlier work (Fickenscher, K. et al. (1987) Eur. J. Biochem. 168, 653-658) the total sequence is not known to an extent of 78%. Comparison with the sequence of the corn NADP-malate dehydrogenase deduced from its cDNA (Metzler, M.C. et al. (1989) Plant Mol. Biol. 12, 713-722) showed 84% agreement; however, the 11 N-terminal residues exhibit only 27% similarity. The N- and C-terminal extrapeptides of the pea NADP-malate dehydrogenase when aligned with non-regulatory NAD-malate dehydrogenases from bacteria or mammals consist of 30 and 17 amino acids, respectively. Since all cysteine-containing peptides were sequenced, the number of eight cysteines per subunit of the pea enzyme was established. The native, oxidized enzyme is characterized by an extremely slow reactivity of two thiols. Titration of the thiols of the denatured, oxidized enzyme both with DTNB and with pCMB resulted in six thiols not involved in disulfide formation. Therefore, one disulfide bridge must be present per 38.9 kDa subunit. Analysis of disulfide bonds by urea gel electrophoresis confirmed this finding. Using digestion products of NADP-malate dehydrogenase with aminopeptidase K, the location of the single disulfide bridge was established to be on the N-terminal arm (Cys-12 and Cys-17) of the polypeptide chain.
Scheibe
R
R
Lehrstuhl für Pflanzenphysiologie, Universität Osnabrück, F.R.G.
Kampfenkel
K
K
Wessels
R
R
Tripier
D
D
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000201:enzymology*; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D050538:Malate Dehydrogenase (NADP+); D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010946:Plants, Medicinal; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology
D004220:Disulfides; D010455:Peptides; D013438:Sulfhydryl Compounds; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+); D014357:Trypsin