Brown E D, Yada R Y
Department of Food Science, University of Guelph, Canada.
Biochim Biophys Acta. 1991 Feb 15;1076(3):406-15. doi: 10.1016/0167-4838(91)90484-h.
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.
以内生硫黄菌和米黑根毛霉天冬氨酸蛋白酶的酶活性及紫外吸收作为变性指标，对其变性过程进行了动力学和平衡分析。结果表明，这些蛋白酶的变性是不可逆的，这表明这些天冬氨酸蛋白酶的构象可能在其酶原中就已预先确定。这些蛋白酶的热变性和盐酸胍变性呈现出一级、两态的动力学行为。这些蛋白酶的平衡去折叠转变具有高度协同性，但在所采用的两个指标中并不完全一致，这表明其与两态特征存在一定偏差。用偏高碘酸钠氧化去除米黑根毛霉糖蛋白酶37.8%的碳水化合物后，其动力学稳定性和平衡稳定性均大幅下降，而氨基酸组成和比活性未发生改变。此外，平衡研究后的凝胶过滤表明，米黑根毛霉蛋白酶部分去除碳水化合物会促进其在变性条件下的自溶。
