The thermal denaturation of Escherichia coli glucosamine-6-phosphate deaminase (G6PD) at neutral pH was studied by means of differential scanning calorimetry (DSC). In the concentration range 0.6-7.3 mg mL-1, the denaturation of this hexameric enzyme was completely irreversible as judged by the absence of any endotherm on rescanning of previously scanned samples. However, the study of the effect of scanning rate on DSC curves indicated that the denaturation of G6PD is, most likely, a complex process which includes transitions in equilibrium as well as an irreversible step; in addition, it was found that application of the equilibrium formalism to the analysis of calorimetric data seems to be justified in this case, provided that scanning rates used are above 0.75 K min-1. The calorimetric and van't Hoff enthalpies for G6PD were 1260 +/- 118 and 160 +/- 27 kcal mol-1, respectively, indicating the presence of intermediates in the process. Accordingly, the DSC curves were adequately fitted to a model including six two-state sequential transitions. The observed protein-concentration dependence of the temperature at the maximum heat capacity, Tm, for each of the individual transitions suggests that G6PD dissociates to dimers in two consecutive steps. Using a model that includes dissociation explicitly, we calculated the thermodynamic parameters for each step. From this data, the enthalpy and free energy for the disruption of one dimer-dimer contact were roughly estimated, at pH 7.1 and 51 degrees C, as 57 and 2.1 kcal mol-1, respectively. Study holds ProTherm entries: 4547 Extra Details: glucosamine-6-phosphate deaminase; hexameric enzyme; endotherm;,scanning rate; intermediates; thermodynamic parameters
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:26 p.m.
|Number of data points||3|
|Proteins||Glucosamine-6-phosphate deaminase ; Glucosamine-6-phosphate deaminase|
|Assays/Quantities/Protocols||Experimental Assay: dHcal ; Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence MRLIPLTTAEQVGKWAARHIVNRINAFKPTADRPFVLGLPTGGTPMTTYKALVEMHKAGQVSFKHVVTFNMDEYVGLPKEHPESYYSFMHRNFFDHVDIPAENINLLNGNAPDIDAECRQYEEKIRSYGKIHLFMGGVGNDGHIAFNEPASSLASRTRIKTLTHDTRVANSRFFDNDVNQVPKYALTVGVGTLLDAEEVMILVLGSQKALALQAAVEGCVNHMWTISCLQLHPKAIMVCDEPSTMELKVKTLRYFNELEAENIKGL|