Equilibrium unfolding studies of the rat liver methionine adenosyltransferase III, a dimeric enzyme with intersubunit active sites.
The reversible unfolding of rat liver methionine adenosyltransferase dimer by urea under equilibrium conditions has been monitored by fluorescence spectroscopy, CD, size-exclusion chromatography, analytical ultracentrifugation and enzyme activity measurements. The results obtained indicate that unfolding takes place through a three-state mechanism, involving an inactive monomeric intermediate. This intermediate has a 70% native secondary structure, binds less 8-anilinonaphthalene-1-sulphonic acid than the native dimer and has a sedimentation coefficient of 4.24+/-0.15. The variations of free energy in the absence of denaturant [DeltaG(H(2)O)] and its coefficients of urea dependence (m), calculated by the linear extrapolation model, were 36.15+/-2.3 kJ.mol(-1) and 19.87+/-0.71 kJ.mol(-1).M(-1) for the dissociation of the native dimer and 14.77+/-1.63 kJ.mol(-1) and 5.23+/-0.21 kJ.mol(-1).M(-1) for the unfolding of the monomeric intermediate respectively. Thus the global free energy change in the absence of denaturant and the m coefficient were calculated to be 65.69 kJ.mol(-1) and 30.33 kJ.mol(-1).M(-1) respectively. Analysis of the calculated thermodynamical parameters indicate the instability of the dimer in the presence of denaturant, and that the major exposure to the solvent is due to dimer dissociation. Finally, a minimum-folding mechanism for methionine adenosyltransferase III is established.
Gasset
María
M
Instituto de Química-Física "Rocasolano" (CSIC), Serrano 119, 28006 Madrid, Spain.
Alfonso
Carlos
C
Neira
José L
JL
Rivas
Germán
G
Pajares
María A
MA
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019281:Dimerization; D005456:Fluorescent Dyes; D008099:Liver / Q000201:enzymology*; D008716:Methionine Adenosyltransferase / Q000737:chemistry / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D051381:Rats; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D008716:Methionine Adenosyltransferase