Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G2W1.
Arch Biochem Biophys. 2011 Sep 15;513(2):102-9. doi: 10.1016/j.abb.2011.06.017. Epub 2011 Jul 13.
Plasmepsin II (PMII), an aspartic protease from the malarial parasite Plasmodium falciparum, represents a model for understanding protease structure/function relationships due to its unique structure and properties. The present study undertook a thermodynamic and kinetic analysis of the PMII folding mechanism and a pH stability profile. Differential scanning calorimetry revealed that the native state of PMII (Np) was irreversibly unfolded, and in the pH range of 6.5-8.0, PMII refolds to a denatured state (Rp) with higher thermal stability than Np. Rp could also be formed upon partially unfolding PMII at pH 11.0 and 37 °C for 2h, followed by adjustment to a pH in the range of 6.5-8.0. While Rp could be folded/unfolded reversibly, Np was shown to exist as a kinetically trapped state. By examining the unfolding kinetics of Np and the kinetics of Rp folding to Np at 25 °C, it was found that Np is kinetically trapped by an unfolding barrier of 25.5 kcal/mol, and yet once unfolded, is prevented from folding by a comparable folding barrier. The folding mechanism of PMII is similar to that reported for pepsin. It is hypothesized that the PMII zymogen also utilizes a prosegment-catalyzed folding mechanism.
疟原虫裂殖子的天冬氨酸蛋白酶 Plasmepsin II（PMII），因其独特的结构和特性，成为研究蛋白酶结构与功能关系的模式酶。本研究对 PMII 的折叠机制和 pH 稳定性进行了热力学和动力学分析。差示扫描量热法显示，PMII 的天然状态（Np）是不可逆展开的，在 pH 值为 6.5-8.0 的范围内，PMII 重新折叠成变性状态（Rp），其热稳定性高于 Np。在 pH 值为 11.0 和 37°C 下部分展开 PMII 2 小时后，也可以形成 Rp，然后将 pH 值调整到 6.5-8.0 的范围内。虽然 Rp 可以可逆折叠/展开，但 Np 表现为动力学捕获状态。通过研究 Np 的展开动力学和 Rp 折叠为 Np 的动力学，在 25°C 下，发现 Np 被 25.5 kcal/mol 的展开势垒动力学捕获，但一旦展开，就会被相当的折叠势垒阻止折叠。PMII 的折叠机制与胃蛋白酶相似。推测 PMII 酶原也利用前肽催化的折叠机制。
