Stability of subtilisins and related proteinases (subtilases).
The stability towards thermal and chemical (guanidine hydrochloride, GnHCl) denaturation of six inhibited subtilases (mesentericopeptidase, subtilisins BPN', Carlsberg and DY, proteinase K and thermitase) has been investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroic and fluorescence spectroscopy. Experiments in the absence and presence of extraneous calcium in the concentration range 2 x 10(-3)-10(-1) M were performed. The presence of calcium in the weak calcium binding site changes the denaturation drastically. The heat- (or GnHCl-) induced unfolding curves obtained using CD spectroscopy show two independent transitions which seem not to have been resolved before. The presence of Ca2+ in the second (third in the case of thermitase) binding site increases the Tm values by 11-21 degrees C and the delta GD(H2O) values obtained from denaturation experiments in GnHCl by 6.7-7.2 kcal/mol when an extraneous Ca2+ concentration of 2 x 10(-2) M was used. One interpretation is that the initial step of denaturation in the presence of added calcium is the formation of a partially unfolded intermediate form, retaining a highly ordered structure with 60-85% of the alpha-helix structure of the native enzyme. This intermediate then unfolds at a temperature considerably higher than that of the same proteinases in the absence of added Ca2+. The free energy of stabilization of the intermediates is increased by 1.8-2.8 times in comparison with that for the unfolding reactions of the subtilases with empty Ca2/Ca3 binding sites. A second interpretation is that the two steps in the unfolding curves correspond to enzyme without and with calcium in the weak binding site. Fluorescence experiments confirm the mechanism involving the formation of intermediate states. The results are discussed in relation to the X-ray models of the six subtilases.
Genov
N
N
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia.
Filippi
B
B
Dolashka
P
P
Wilson
K S
KS
Betzel
C
C
D001665:Binding Sites; D002118:Calcium / Q000032:analysis / Q000494:pharmacology; D002942:Circular Dichroism; D019286:Endopeptidase K; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D011489:Protein Denaturation; D017510:Protein Folding; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
D006146:Guanidines; D012697:Serine Endopeptidases; D013381:Subtilisins; C021553:mesentericopeptidase; C022728:thermitase; D019286:Endopeptidase K; D019791:Guanidine; D002118:Calcium