NADP(+)-malic enzyme from sugarcane leaves: structural properties studied by thermal inactivation.
The irreversible thermal inactivation of the sugarcane leaf NADP(+)-malic enzyme was studied at 50 degrees C and pH 7.0 and 8.0. Depending on the preincubation conditions, thermal inactivation followed mono- or biphasic first-order kinetics. A two-step behavior in the irreversible denaturation process was found when protein concentration was sufficiently low. The protein concentration necessary to obtain monlphasic thermal inactivation kinetics was lower at pH 8.0 than at pH 7.0. The results suggest that biphasic inactivation kinetics are the consequence of the existence of two different oligomeric forms of the enzyme (dimer and tetramer), with the dimer being more stable in regards to thermal inactivation. The effects of the substrate and essential cofactors on the thermostability and equilibrium between the dimeric and tetrameric enzyme forms were also studied. Depending on the pH, NADP+, L-malate, and Mg2+ all had a protective effect on the stability of the dimeric and tetrameric species during thermal treatment. However, these ligands showed different effects on the aggregation state of the enzyme. NADP+ and L-malate induced dissociation, especially at pH 8.0, whereas Mg2+ induced aggregation of the protein. By studying the thermal inactivation kinetics at 50 degrees C and different pH values it was observed that the equilibrium between dimers and tetramers was dramatically affected in the range of pH 7.0-8.0. These results suggest that an amino acid residue(s) in the protein with an apparent pKa value of 7.7 needs to be deprotonated to stabilize aggregation of the enzyme to the tetrameric form.
Centro de Estudios Fotosintéticos y Bioquímicos (CONICET, Fundación M. Lillo, UNR), Rosario, Argentina.
A A
AA
Iglesias
C P
CP
Spampinato
C S
CS
Andreo
D004789:Enzyme Activation / Q000187:drug effects; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008291:Malate Dehydrogenase / Q000737:chemistry*; D008293:Malates / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D010940:Plant Proteins / Q000737:chemistry*; D010945:Plants, Edible / Q000201:enzymology*; D011487:Protein Conformation; D013379:Substrate Specificity
D008293:Malates; D010940:Plant Proteins; D009249:NADP; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+); D008274:Magnesium