NADP malic Enzyme from Sugarcane Leaves. Structural Properties Studied by Thermal Inactivation

A.A. IGLESIAS, C.P. SPAMPINATO, C.S. ANDREO

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Año: 1991 vol. 290 p. 272 - 276

0003-9861

The irreversible thermal inactivation of the sugarcaneleaf NADP+-malic enzyme was studied at 50°C and pH‘7.0 and 8.0. Depending on the preincubation conditions,thermal inactivation followed mono- or biphasic firstorderkinetics. A two-step behavior in the irreversibledenaturation process was found when protein concentrationwas sufficiently low. The protein concentrationnecessary to obtain monophasic thermal inactivation kineticswas lower at pH 8.0 than at pH 7.0. The resultssuggest that biphasic inactivation kinetics are the consequenceof the existence of two different oligomericforms of the enzyme (dimer and tetramer), with the dimerbeing more stable in regards to thermal inactivation. Theeffects of the substrate and essential cofactors on thethermostability and equilibrium between the dimeric andtetrameric enzyme forms were also studied. Dependingon the pH, NADP+, L-malate, and Mg2+ all had a protectiveeffect on the stability of the dimeric and tetramericspecies during thermal treatment. However, these ligandsshowed different effects on the aggregation state of theenzyme. NADP+ and L-malate induced dissociation, especiallyat pH 8.0, whereas Mg2+ induced aggregationof the protein. By studying the thermal inactivation kineticsat 50°C and different pH values it was observedthat the equilibrium between dimers and tetramers wasdramatically affected in the range of pH 7.0-8.0. Theseresults suggest that an amino acid residue(s) in the proteinwith an apparent pK,, value of 7.7 needs to be deprotonatedto stabilize aggregation of the enzyme to the tetrameritform. 0 1991 Academic Press, Inc.