Structural and catalytic properties of oxidized and reduced chloroplast NADP-malate dehydrogenase upon denaturation and renaturation.
Chloroplast NADP-dependent malate dehydrogenase exists in two interconvertible forms: the inactive disulfide-containing form and the active dithiol form. No major difference in secondary structure or conformation was found between the oxidized and the reduced enzyme as determined by circular dichroism and intrinsic protein fluorescence. The guanidine/HCl-dependent unfolding of the enzyme is characterized by two transition midpoints: those of the reduced enzyme are lower by about 0.2 M guanidine/HCl compared to the oxidized enzyme. As shown by analytical ultracentrifugation, there was no effect of guanidine/HCl concentrations up to 0.25 M on the quaternary structure of the enzyme in its oxidized and reduced forms: both sedimentation coefficient (S20,w = 4.9 +/- 0.1 S) and sedimentation equilibrium (75 +/- 3 kDa) yield the dimer. In the oxidized state the enzyme undergoes guanidine-dependent dissociation to the monomer with a midpoint of transition at 0.5 M. The kinetics of unfolding were found to be significantly faster for the reduced than for the oxidized enzyme. Renaturation and reactivation of reduced enzyme was more rapid and occurred with higher yields (100%) than for the oxidized enzyme (60-80% yield). Furthermore, the effect of denaturants on catalytic activity, and reductive activation of the oxidized form, were studied. Both increase in protein fluorescence and a stimulatory effect on the activities at low guanidine/HCl concentrations were observed for the oxidized and the reduced form of the enzyme. Denaturants increase the rate of reductive activation of NADP-malate dehydrogenase.
Scheibe
R
R
Lehrstuhl Pflanzenphysiologie, Universität Bayreuth, Federal Republic of Germany.
Rudolph
R
R
Reng
W
W
Jaenicke
R
R
D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D002942:Circular Dichroism; D004220:Disulfides; D004789:Enzyme Activation; D007887:Fabaceae / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000302:isolation & purification*; D050538:Malate Dehydrogenase (NADP+); D010084:Oxidation-Reduction; D010946:Plants, Medicinal; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds
D004220:Disulfides; D013438:Sulfhydryl Compounds; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+)