Determination of the regulatory disulfide bonds of NADP-dependent malate dehydrogenase from Pisum sativum by site-directed mutagenesis.
The light-mediated reversible activation of NADP-dependent malate dehydrogenase (NADP-MDH) from Pisum sativum can be simulated in vitro by reducing the inactive oxidized enzyme with dithiothreitol. Since the gross structure and the dimeric state of the enzyme are unaffected by the state of oxidation, the redox modulation cannot be attributed to inter-subunit disulfide bridges. In order to identify intra-chain cystine cross bridges that might be candidates responsible for the activation reaction, site-directed mutagenesis experiments were performed, substituting alanine for up to four exposed cysteine residues. Mutants were expressed in freshly transformed EcoB cells and purified to homogeneity. As indicated by the activation behavior (by dithiothreitol-mediated thioldisulfide exchange), disulfides C23-C28 in the N-terminal and C364-C376 in the C-terminal part of the polypeptide chain are involved in the light-induced modulation of the activity of the wild type enzyme. A mutant of the enzyme lacking the N-terminal 45 residues confirms this result. Electrophoretic mobility and FPLC prove the wild type enzyme and its mutants to be dimeric; differences refer to the packing of the N- and C-terminal portions of the enzyme in its oxidized and reduced state. The kinetics of the redox modulation differ, depending on the solvent conditions and the mode of activation. After elimination of the N-terminal disulfide bond, sigmoidal activation profiles are no longer observed, suggesting a slow conformational rearrangement in the N-terminal portion of the wild type enzyme to be rate-limiting in the course of reductive activation. For the wild type, this finding can be mimicked in the presence of non-denaturing concentrations of guanidinium-chloride.
Riessland
R
R
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Jaenicke
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R
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D003553:Cystine / Q000032:analysis*; D004220:Disulfides / Q000032:analysis*; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D050538:Malate Dehydrogenase (NADP+); D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D018532:Pisum sativum / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry
D004220:Disulfides; D011994:Recombinant Proteins; D003553:Cystine; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+); D000409:Alanine; D004229:Dithiothreitol