Kinetic and equilibrium analyses of the denaturation of Endothia parasitica and Mucor miehei aspartic proteinases were performed using enzyme activity and ultraviolet absorption as indices of denaturation. Denaturation of these proteinases was shown to be irreversible, suggesting that the conformations of these aspartic proteinases may be predetermined in their zymogens. Thermal and guanidine hydrochloride denaturation of these proteinases produced first-order, two-state, kinetic behaviour. Equilibrium unfolding transitions of these proteinases were highly cooperative but not entirely coincident in the two indices employed, suggesting some deviation from two-state character. Oxidation to remove 37.8% of the carbohydrate of M. miehei glycoproteinase with sodium metaperiodate resulted in a substantial decrease in both kinetic and equilibrium stabilities without modification of the amino acid composition or specific activity. In addition, gel filtration subsequent to equilibrium studies indicated that partial removal of the carbohydrate from M. miehei proteinase promoted autolysis under denaturing conditions. Study holds ProTherm entries: 10030, 10031, 10032, 10033 Extra Details: aspartic proteinase; protein denaturation; kinetics; (E.parasitica);,(M.miehei)
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:39 p.m.
|Number of data points
|Mucorpepsin ; Mucorpepsin
|Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O
|Mutations for sequence MLFSQITSAILLTAASLSLTTARPVSKQSESKDKLLALPLTSVSRKFSQTKFGQQQLAEKLAGLKPFSEAAADGSVDTPGYYDFDLEEYAIPVSIGTPGQDFLLLFDTGSSDTWVPHKGCTKSEGCVGSRFFDPSASSTFKATNYNLNITYGTGGANGLYFEDSIAIGDITVTKQILAYVDNVRGPTAEQSPNADIFLDGLFGAAYPDNTAMEAEYGSTYNTVHVNLYKQGLISSPLFSVYMNTNSGTGEVVFGGVNNTLLGGDIAYTDVMSRYGGYYFWDAPVTGITVDGSAAVRFSRPQAFTIDTGTNFFIMPSSAASKIVKAALPDATETQQGWVVPCASYQNSKSTISIVMQKSGSSSDTIEISVPVSKMLLPVDQSNETCMFIILPDGGNQYIVGNLFLRFFVNVYDFGNNRIGFAPLASAYENE ; Mutations for sequence AAADGSVDTPGYYDFDLEEYAIPVSIGTPGQDFLLLFDTGSSDTWVPHKGCTKSEGCVGSRFFDPSASSTFKATNYNLNITYGTGGANGLYFEDSIAIGDITVTKQILAYVDNVRGPTAEQSPNADIFLDGLFGAAYPDNTAMEAEYGSTYNTVHVNLYKQGLISSPLFSVYMNTNSGTGEVVFGGVNNTLLGGDIAYTDVMSRYGGYYFWDAPVTGITVDGSAAVRFSRPQAFTIDTGTNFFIMPSSAASKIVKAALPDATETQQGWVVPCASYQNSKSTISIVMQKSGSSSDTIEISVPVSKMLLPVDQSNETCMFIILPDGGNQYIVGNLFLRFFVNVYDFGNNRIGFAPLASAYENE