Spectroscopic studies on proteinase K and subtilisin DY. Relation to X-ray models.
Circular dichroic spectroscopy has been used to study the effect of pH, guanidinium hydrochloride concentration and temperature on the conformation of the fungal subtilisin-like proteinase K and the bacterial DY. The ellipticity of the bands in the far ultraviolet region remains almost unchanged in the pH range 3.0-11.0 (PMS-proteinase K) and 5.0-10.0 (PMS-subtilisin DY). The same ranges of pH stability were determined from the pH dependence of the near ultraviolet dichroic spectra. Hence the changes in the tertiary and secondary structure occur in parallel. Proteinase K is considerably more stable at acidic and somewhat more stable at alkaline pH than subtilisin DY. At neutral pH proteinase K is more resistant to denaturation by guanidinium hydrochloride than is subtilisin DY. The midpoints of the denaturation curves were 6.2 M and 3.2 M guanidinium, respectively. The thermal unfolding of proteinase K occurred at a higher temperature than for subtilisin DY, the transition midpoints being 65 degrees and 48 degrees, respectively. Thus proteinase K is overall a much more robust molecule than subtilisin DY, showing greater resistance to all three forms of denaturation. The differences in the stability of the two proteinases can be partly explained by differences in their calcium binding sites.
Dolashka
P
P
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia.
Filippi
B
B
Wilson
K S
KS
Betzel
C
C
Genov
N
N
D002942:Circular Dichroism; D019286:Endopeptidase K; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000187:drug effects; D013056:Spectrophotometry, Ultraviolet; D013381:Subtilisins / Q000737:chemistry* / Q000187:drug effects
D006146:Guanidines; D012697:Serine Endopeptidases; D013381:Subtilisins; D019286:Endopeptidase K; D019791:Guanidine