An irreversible and kinetically controlled process: thermal induced denaturation of L-2-hydroxyisocaproate dehydrogenase from Lactobacillus confusus.


Abstract

The thermal denaturation of Lactobacillus confusus L-2-Hydroxyisocaproate Dehydrogenase (L-HicDH) has been studied by Differential Scanning Calorimetry (DSC). The stability of this enzyme has been investigated at different pH conditions. The results of this study indicate that the thermal denaturation of this enzyme is irreversible and the T(m) is dependent on the scan-rate, which suggests that the denaturation process of L-HicDH is kinetically determined. The heat capacity function of L-HicDH shows a single peak with the T(m) values between 52.14 degrees C and 55.89 degrees C at pH 7.0 at different scan rates. These results indicate that the whole L-HicDH could unfold as a single cooperative unit, and intersubunit interactions of this homotetrameric enzyme must play a significant role in the stabilization of the whole enzyme. The rate constant of the unfolding is analyzed as a first order kinetic constant with the Arrhenius equation, and the activation energy has been calculated. The variation of the activation energy values obtained with different methods does not support the validity of the one-step irreversible model. The denaturation pathway was described by a three-state model, N --> U --> F, in which the dissociation of the tetramer takes place as an irreversible step before the irreversible unfolding of the monomers. The calorimetric enthalpy associated with the irreversible dissociation and the calorimetric enthalpy associated with the unfolding of the monomer were obtained from the best fitting procedure. Thermal unfolding of L-HicDH was also studied using Circular Dichroism (CD) spectroscopy. Both methods yielded comparable values. Study holds ProTherm entries: 23918, 23919, 23920, 23921, 23922, 23923, 23924, 23925, 23926, 23927, 23928, 23929, 23930, 23931, 23932, 23933, 23934 Extra Details: scan rate: 1 K/min L-HicDH; Differential Scanning Calorimetry (DSC); thermal denaturation; transition temperature; enthalpy; activation energy

Submission Details

ID: 4zCYsjTA4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:54 p.m.

Version: 1

Publication Details
Bao L;Chatterjee S;Lohmer S;Schomburg D,Protein J. (2007) An irreversible and kinetically controlled process: thermal induced denaturation of L-2-hydroxyisocaproate dehydrogenase from Lactobacillus confusus. PMID:17205397
Additional Information

Number of data points 31
Proteins L-2-hydroxyisocaproate dehydrogenase ; L-2-hydroxyisocaproate dehydrogenase
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: Tm buffers:Potassium phosphate: 5 mM, ionic:: ; Experimental Assay: Tm buffers:Potassium phosphate: 5 mM, ionic:: , pH:8.0 ; Experimental Assay: Tm buffers:Potassium phosphate: 5 mM, ionic:: , pH:7.0 ; Experimental Assay: Tm buffers:Potassium phosphate: 5 mM, ionic:: , prot_conc:0.2 mg/mL ; Experimental Assay: Tm buffers:Potassium phosphate: 5 mM, ionic:: , prot_conc:0.2 mg/mL, pH:8.0 ; Experimental Assay: Tm buffers:Potassium phosphate: 5 mM, ionic:: , prot_conc:0.2 mg/mL, pH:7.0 ; Experimental Assay: dHcal pH:8.0 ; Experimental Assay: Tm buffers:Natrium phosphate, Imidazol: 50 mM, 250 mM, ionic:NaCl: 300 mM, pH:8.0 ; Experimental Assay: dHvH pH:8.0 ; Experimental Assay: dHcal pH:7.0 ; Experimental Assay: Tm buffers:Natrium phosphate, Imidazol: 50 mM, 250 mM, ionic:NaCl: 300 mM, pH:7.0 ; Experimental Assay: dHvH pH:7.0 ; Experimental Assay: dHcal pH:6.0 ; Experimental Assay: Tm buffers:Natrium phosphate, Imidazol: 50 mM, 250 mM, ionic:NaCl: 300 mM ; Experimental Assay: dHvH pH:6.0
Libraries Mutations for sequence ARKIGIIGLGNVGAAVAHGLIAQGVADDYVFIDANEAKVKADQIDFQDAMANLEAHGNIVINDWAALADADVVISTLGNIKLQQDNPTGDRFAELKFTSSMVQSVGTNLKESGFHGVLVVISNPVDVITALFQHVTGFPAHKVIGTGTLLDTARMQRAVGEAFDLDPRSVSGYNLGEHGNSQFVAWSTVRVMGQPIVTLADAGDIDLAAIEEEARKGGFTVLNGKGYTSYGVATSAIRIAKAVMADAHAELVVSNRRDDMGMYLSYPAIIGRDGVLAETTLDLTTDEQEKLLQSRDYIQQRFDEIVDTL
Sequence Assay Result Units